Antibiotic Resistance Mechanisms in Gram-Negative Bacteria

CHARACTERISATION OF ANTIBIOTIC RESISTANCE
MECHANISMS IN GRAM-NEGATIVE BACTERIA FROM

TRIPOLI AND BENGHAZI, LIBYA
By
Allaaeddin Ali El Salabi
A thesis submitted for the degree of Doctor of Philosophy

at Cardiff University
2011
Cardiff University, School of Medicine, Department of

Infection, Immunity & Biochemistry
UMI Number: U585511
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DECLARATION
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not concurrently submitted in candidate for any degree.
S ig n e d ^ r r ly /f ^ ^ ? (candidate) Date .
STATEMENT 1
This thesis is being submitted in partial fulfilment of the requirements for the
degree of PhD
"i^m d ^ ^ / (candidate) Date
STATEMENT 2
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where otherwise stated. Other sources are acknowledged by explicit references
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ii
Summary
As very little information is known of the antibiotic resistance in Gram-
negative bacteria in Libya in addition to the desperate need for insight
knowledge o f the antibiotic resistance in Libyan hospitals, this study was
undertaken to investigate the mechanism of antibiotic resistance in isolates
collected from clinical, non-clinical and environmental samples from Tripoli
and Benghazi, Libya. Bacterial collection include samples taken from patients
admitted to the hospitals in ICUs and other wards, they also include swabs
randomly collected from hospitals environment. These swabs were from walls,
bedsides, curtains, floors, toilets, workstations, mechanical ventilators,
stainless steel containers and instruments used in particular ICUs. This study
clearly demonstrates the emergence of MDR Gram-negative bacteria in
Tripoli and Benghazi hospitals, these MDR bacteria were clinical and non-
clinical revealing the long standing infection control problem in these
hospitals. K. pneumoniae was found as the most frequently isolated strain
being disseminated in hospitals and outside hospitals followed by E. coli. K.
pneumoniae and E. coli were detected harbouring blactx-m group 1 in
association with ISEcpl the enhancer of the p-lactamase gene movement.
More importantly, ^/«ctx-m-is in association with ISEcpl were detected carried
on conjugative plasmids o f different sizes and able to move via Libyan K.
pneumoniae and E. coli to sensitive bacteria via conjugation. Some isolates of
K. pneumoniae were clonally related and were in some cases found in

different hospital revealing the outbreak of MDR K. pneumoniae in Libyan
hospitals. E. coli strains showed the emergence o f more than one clone in one
hospital which indicates to the lack of hospital hygiene. Three novel sequence
types among K. pneumoniae were discovered in this study, one of which K.
pneumoniae AES817 that assigned ST511 was collected from one of Benghazi
streets and was found carrying blacrx-M-15 and ISEcpl on a plasmid of 400kb.
Characterisation o f P. aeruginosa showed the emergence of clonally related
strains carrying blaym-2, one was isolated from a patient admitted to Al-Jalla
hospital in Benghazi and the other from a stainless steel container from the
same hospital but different ward, this MBL was found on a novel integron in
both strains. Interestingly, 6 /<zvim-2 was found chromosomally mediated
proposing that the dissemination o f this MBL might be due to mobile genetic
elements. Perhaps the most interesting finding o f this study is 6/<3tmb-i which
was detected in environmental strain swabbed from the floor of Tripoli central
hospital. This MBL was unusual in terms of the similarity this gene shares
with other known MBLs and also to the discovery of this MBL carried by
environmental bacteria A. xylosoxidans, it is moreover the first MBL
discovered in Libya.
Presentations and Publications
Presentations given from this study
1- Phenotypic and Genotypic Characterisation of Clinical and non-

Clinical Gram-negative Bacteria from Benghazi-Libya. 49th
Interscience Conference on Antimicrobial Agents and Chemotherapy,
September 12-15, 2009, San Francisco.
2- Identification o f Tn402, Class 1 integrons and ISCR elements among

endemic multi-drug-resistant Klebsiella pneumoniae from Benghazi-
Libya. 49th Interscience Conference on Antimicrobial Agents and
Chemotherapy, September 12-15, 2009, San Francisco.
3- Novel subclass o f a Group B1 Metallo-p-lactamase, bla-\uB-u in

Clinical and non Clinical Gram-negative Bacteria from Libya. 49th
Interscience Conference on Antimicrobial Agents and Chemotherapy,
September 12-15, 2009, San Francisco.
4- The tniC-like transposon Tn5090 is commonly found in Klebsiella

pneumoniae isolates from Portugal and North Africa. 49th Interscience
Conference on Antimicrobial Agents and Chemotherapy, September
12-15, 2009, San Francisco.
V
Publications and publications in collaborations
1- Salabi, A. E., M. A. Toleman, J. Weeks, T. Bruderer, R. Frei, and T. R.

Walsh. 2010. First report of the metallo-beta-lactamase SPM-1 in
Europe. Antimicrob Agents Chemother 54:582.
2- Chouchani, C., R. Marrakchi, and A. El Salabi. 2011. Evolution of beta-

lactams resistance in Gram-negative bacteria in Tunisia. Crit Rev
Microbiol 37:167-177.
3- Chouchani, C., R. Marrakchi, L. Ferchichi, A. El Salabi, and T. R.

Walsh. 2011. VIM and IMP metallo-beta-lactamases and other extended-
spectrum beta-lactamases in Escherichia coli and Klebsiella pneumoniae
from environmental samples in a Tunisian hospital. Apmis 119:725-732.
4- Chouchani, C., A. El Salabi, R. Marrakchi, L. Ferchichi, and T. R.

Walsh.Characterization o f IncA/C conjugative plasmid harbouring
blajEM-52 and blacrx-u-is extended-spectrum p-lactamases in clinical
isolates o f Escherichia coli in Tunisia (accepted).
5- Allaaeddin El Salabi, Pardha Saradhi Borra, Mark A. Toleman, 0rjan

Samuelsen and Timothy R. Walsh Genetic and biochemical
characterization o f a novel metallo-p-lactamase, TMB-1, from a
Achromobacter xylosoxidans strain isolated from Tripoli, Libya
(submitted)
6- Allaaeddin El Salabi, Mark A. Toleman, Ahmed Matmati, Chedly

Chouchani and Timothy R. Walsh blawu -2 positive Pseudomonas
aeruginosa isolated from operating apparatus and patients in Tripoli,
Libya (submitted)
7- Allaaeddin El Salabi, Mark A. Toleman, Abdulazizi Zorgani and

Timothy R. Walsh. Molecular characterization of antibiotic resistance
mechanisms in K. pneumoniae isolated from Tripoli and Benghazi
hospitals (in progress)
8- Allaaeddin El Salabi, Mark A. Toleman, Asma Alramli and Timothy R.

Walsh. Molecular characterization of antibiotic resistance mechanisms
in E. coli collected from Tripoli and Benghazi hospitals (in progress)
vi
Acknowledgements
My thanks go first and foremost to Allah, who created me to discover how great he is and
increase my faith in him.
I would like to thank the following people without whose help the completion o f this thesis
would not have been possible.
To my supervisor Prof Timothy Rutland Walsh, who taught me how innovation could be
achieved and continued to support and encourage the spirit o f scientific discovery in me.
To my advisor Dr Mark Alexander Toleman for his valuable advice and support, who taught
me the molecular genetics o f bacteria.
To Mrs Janis Weeks for her unending help and support, with a smile if nothing else.
To the staff at Reference Centre for Detection o f Antimicrobial Resistance, Department of

Microbiology and Infection Control, University Hospital o f North Norway for their valuable
advice and help with the TMB-1 purification.
To Dr Henry Ryley for his help with the PFGE pictures analysis.
To Dr Amanda Tonks for her encouragement and support
To Dr Mandy Wootton and staff at her laboratory for their valuable help with the
identification o f Libyan isolates
To all staff within section o f Medical Microbiology, School o f Medicine, University hospital
o f Wales for their lovely support
To my favourite brother Fakhrieddin El Salabi, who was waiting to share with me the
moments o f completion my studies and passed away before the completion o f my PhD.
To all my family members for their irrepressible support and encouragement with which I
could not have even begun this.
To Prof Salha Ben-Gwirif for her encouragement and support and the spirit of Dr Fathi
Belied, who taught me the basics o f microbiology.
To my friend Ahmed Gawhari and his wife Neven, for their lovely support and help.
To my friend Othman Boaisha for his kind encouragement and support
To staff o f Faculty o f Public Health, University o f Benghazi for their support
To Ranya, my wife for, her support, patience and endurance
Finally to Amina Benour my mother, whom I love, who supported me, prayed for me, looked
after me and was very close to me even at a distance, her help and encouragement was
unbelievable.
This thesis is dedicated to
the spirit o f my father, the
spirit o f my brother and
to the new Libya

LIST OF FIGURES
Fig. 1.1 Chemical structure o f daptomycin.............................................. 6
Fig. 1 .2 Chemical structure o f oxazolidinone radezolid................... 6
Fig. 1.3 Chemical structure o f the fluoroquinolone delafloxacin 6
Fig. 1.4 Chemical structure o f the aminoglycoside ACHN-490 ....... 7
Fig. 1.5 Chemical structure o f the tetracycline om adacycline 7
Fig. 1.6 Chemical structure o f the Avibactam N X L -104....................... 7
Fig. 1.7 Cell wall envelopes o f Gram-positive and Gram-negative
bacteria.................................................................................... 8
Fig. 1.8 N-formimodoyl-thienamycin............................................... 15
Fig. 1.9 Inhibition o f protein synthesis by aminoglycosides 19
Fig. l.li Inhibition o f cell wall synthesis by p-lactam s.................... 20
Fig. 1.1 Inhibition o f DNA synthesis by quinolones........................ 20
Fig. l.i: Global emergence o f M B L s................................................... 32
Fig. l.i: The emergence o f CTX-M type ESBLs in E urope 33
Fig. 1.1- The occurrence o f K. pneumoniae resistant to 3rd generation
cephalosporins in Europe.................................................. 36
Fig. 1.1: The occurrence o f P. aeruginosa resistant to carbapenems
in E urope.................................................................................. 37
Fig. 1.16 Annual rate o f antimicrobial resistance among E. coli
isolates (4394 isolates) tested against selected agents from
the MYSTIC program ................................................................ 41
Fig. 1.17 Annual rate o f antimicrobial resistance among K.
pneumoniae isolates (2694 isolates) tested against selected
against selected agents from the MYSTIC program................ 41
Fig. 1.18 The dissemination o f P-lactamases, ESBLs and
carbapenemases in T unisia...................................................... 43
Fig. 1.19 Genomic island o f A. baumannii A Y E .................................... 46
Fig. 3.1 Multiplex PCR experiment to detected the incidence of CTX-
M type ESBLs groups 1, 2, 8, 9 and 26................................. 92
Fig. 3.2 PCR experiment to detect the incidence of balcix-u-\s in K.
pneum oniae................................................................................. 92
Fig. 3.3 PCR experiment to detect the incidence of blaax-M group 1 in
association with ISEcpl in K. pneum oniae............................. 93
Fig. 3.4 PCR experiment to detect disrupted ISEcpl sequence in K.
pneum oniae................................................................................ 93
Fig. 3.5 Diagram showing the genetic environment o f blacix-u -15
g e n e ........................................................................................... 94
Fig. 3.6 Blotting o f K. pneumoniae isolates (1-47) and probing with
blajvM .......................................................................................... 95
x
blciTEM ........................................................................................... 96
bla^wy........................................................................................... 96
blasHv............................................................................................. 97
blacTx-M-is/IS/scpf.................................................................... 98
M * c t x -m - 1 5 ....................................................................... 98
Fig. 3.12 PFGE o f *S1 digests o f a subset of K. pneumoniae isolates... 100
Fig. 3.13 Autorad after probing with blacvx-u-\ s/lSEcpl o f blotted
PFGE from fig.3.12.................................................................... 101
Fig. 3.14 K. pneumoniae typed by R A P D ........................................... 104
Fig. 3.15 PFGE o f X ba\ digests o f a subset o f K. pneumoniae isolates 106
Fig. 3.16 PFGE of X ba\ digests o f a subset o f K. pneumoniae isolates 107
Fig. 3.17 Dendrogram of PFGE gel picture from fig. 3 .1 5 .................. 108
Fig. 3.18 Dendrogram o f PFGE gel picture from fig. 3 .1 6 .................. 109
Fig. 3.19 Autorad after probing with blacrx-M-is of blotted PFGE from
fig.3.15........................................................................................ 114
Fig. 3.20 Autorad after probing with 6/<3c t x -m - i 5 o f blotted PFGE from
fig.3.16........................................................................................ 115
Fig. 3.21 Detection o f blacjx-M group 1/lSEcp] in G W E. coli
XI
transconjugants o f K. pneumoniae AES isolates................ 116
Fig. 3.22 Detection of the occurrence of an intact and disrupted copies
o f ISE cpfm GFP transconjugants of K. pneumoniae AES
iso lates...................................................................................... 116
Fig. 3.23 PFGE o f *S1 digests o f K. pneumoniae and GFP E .coli
transconjugants........................................................................ 118
Fig. 3.24 Autorad after probing with blaax-u -\5 of blotted PFGE from
fig. 3 .2 3 ....................................................................................... 119
Fig. 3.25 PFGE o f S \ digests o f K. pneumoniae and GFP E. coli
transconjugants........................................................................... 120
Fig. 3.26 Autorad after probing with blacix-M-isftSEcpl o f blotted
PFGE from fig.3.25............................................................... 121
Fig. 3.27 Amplification o f classical class 1 integron from a subset of
K. pneumoniae............................................................................ 123
Fig. 3.28 Genetic context o f class 1 integrons found in
K. pneum oniae........................................................................... 124
Fig. 3.29 Detection o f transposons among a subset of K. pneumoniae 126
Fig. 3.30 Genetic context o f Tn402transposons found in
K. pneum oniae......................................................................... 126
Fig. 3.31 PFGE o f S \ digests o f a subset of K. pneum oniae............... 128
Fig. 3.32 Autorad after probing with tniC of blotted PFGE from
fig.3.31 ....................................................................................... 128
xii
Fig. 3.33 PFGE o f £1 digests o f a subset o f K. pneum oniae............... 129
Fig. 3.34 Autorad after probing with tniC of blotted PFGE from
fig.3.3 3 ........................................................................................ 129
Fig. 3.35 Probing o f K. pneumoniae isolates (1-47) with ISCR2 gene 130
Fig. 3.36 Probing o f K. pneumoniae isolates (48-80) with ISCR2gene 131
Fig. 4.1 Multiplex PCR to detect CTX-M groups 1, 2, 8 , 9 &26 in
E. coli isolates.............................................................................. 144
Fig. 4.2 Multiplex PCR to detect CTX-M groups 1, 2, 8 , 9 &26 in
E. coli isolates............................................................................... 145
Fig. 4.3 Detection o f ft/acTX-M group 1 and ISEcpl in E. c o li 145
Fig. 4.4 Detection o f blact x - m group 1 and ISEcpl in E. c o li 146
Fig. 4.5 Detection o f blacxx-u group 1 in association with an intact
ISEcpl in E. coli.......................................................................... 146
Fig. 4.6 Detection o f blacrx-u groupl in association with an intact
ISEcpl in E. coli........................................................................... 147
Fig. 4.7 PFGE o f S \ digestion o f E. coli parents and transconjugants 150
Fig. 4.8 Autorad o f E. coli parents and transconjugants after probing
o f PFGE gel from fig. 4.7 with ^/actx-m-i s ............................. 151
Fig. 4.9 PFGE o f S \ digestion o f E. coli parents and transconjugants 152
Fig. 4.10 Autorad o f E. coli parents and transconjugants after probing
o f PFGE gel from fig. 4.9 with blacjx-M-is/ISEcpl 153
Fig. 4.11 PFGE o f Xbal digestion and separation o f a subset of E. coli
Xlll
genomic D N A ............................................................................ 155
Fig. 4.12 Dendrogram o f PFGE gel picture from fig. 4.11 ................ 156
Fig. 4.13 PFGE o f Xbal digestion and separation of a subset of E. coli 157
genomic D N A .............................................................................
Fig. 4.14 Dendrogram o f PFGE gel picture from fig. 4 .1 3 .................... 157
Fig. 4.15 Autorad o f PFGE gel o f fig. 4.11 after probing with
blac t x - m - 1 5 ................................................................................................................................................................................................................................................. 159
Fig. 4.16 Autorad o f PFGE gel o f fig 4.13 after probing with
blac t x - m - 1 5 .............................................................................................................................................................................................................................................. 160
Fig. 4.17 Genetic context o f class 1 integrons found in E. c o li 161
Fig. 5.1 Etest o f P. aeruginosa............................................................... 172
Fig. 5.2 Detection of Tn402, Tn21 and blayim-2 in P. aeruginosa
AES81 and A E S 83...................................................................... 173
Fig. 5.3 PFGE o f Spe-1 digestion of 14 E. coli isolates..................... 175
Fig. 5.4 Dendrogram o f PFGE gel picture of fig. 5 .3 ........................ 176
Fig. 5.5 PFGE o f S 1 digestion o f a subset of P. aeruginosa isolates ... 176
Fig. 5.6 Autorad o f PFGE gel o f fig. 5.5 after probing with blawu-i 177
Fig. 5.7 PFGE o f Spe 1 digestion o f a subset o f P. aeruginosa isolates 178
Fig. 5.8 Autorad of PFGE gel of fig. 5.7 after probing with h/aviM-2 179
Fig. 5.9 Amplification o f class 1 integrons from a subset of
P. aeruginosa............................................................................ 181
Fig. 5.10 Genetic contexts o f class 1 integrons found in P. aeruginosa 182
xiv
Fig. 6.1 Genetic context o f class 1 integrons from A. xylosoxidans 192
Fig. 6.2 Detection o f genetic location of 6/« tm b - i in A. xylosoxidans 194
Fig. 6.3 Dendrogram o f comparison of amino acid sequence o f the
p-lactamase TMB-1 and other acquired M B L s.................... 196
Fig. 6.4 Comparison o f amino acid sequence of p-lactamase TMB-1
and other acquired MBLs......................................................... 197
Fig. 6.5 Secondary structure o f TMB-1 compared to that of VIM-2 198
Fig. 7.1 Map o f Libya 205
XV
LIST OF TABLES
Table 1.1 History o f antibiotic introductions and approval................. 5
Table 1.2 Main classes o f antibiotics and p-lactamase inhibitors 11
Table 1.3 Longitudinal increase in multi-drug resistance in USA 39
Table 2.1 Multiplex PCR primers for CTX-M groups 1, 2, 8 , 9 & 26 64
Table 2.2 Oligonucleotide sequences to detect 6 /<zoxa-48 and IS 1999 67
Table 2.3 Oligonucleotide sequences used for PCR amplification of
housekeeping g e n e s............................................................... 71
Table 3.1 Dissemination o f K. pneumoniae in Tripoli and Benghazi 89
Table 3.2 MIC 50 and MIC90 o f K. pneumoniae....................................... 90
Table 3.3 The incidence o f blacix-u group 1; blaj^M, blasnv and ISCR2 95
Table 4.1 MIC50 and MIC90 o f E. coli isolates....................................... 144
Table 4.2 Sensitivity profile E. coli parents and transconjugants 148
Table 5.1 List o f P. aeruginosa used in experiments.......................... 171
Table 5.2 Antibiotic sensitivity testing of clinical and non-clinical
isolates o f P. aeruginosa........................................................... 1
Table 6 .1 Steady-state kinetic constants of TMB-1 and GIM-1 200
xvi
LIST OF ABBREVIATIONS
ABC ATP binding cassette
AES Allaaeddin El Salabi
AIM-1 Australian imipenemase
AmpC ampicillin
ASP Asparagin
attC attachment site
bla p-lactamase
CAI community acquired infections
CIAI complicated intra-abdominal infection
CR common region
CSSSI complicated skin and skin structure infection
CTX-M Cefotaximase
CUTI complicated urinary tract infection
CVL Cervicovaginal Lavage
Cys Cystein
Dhfr dihydrofolate reductase
DIM Dutch imipenemase
EARSS European antimicrobial resistance surveillance system
EDTA Ethylenediaminetetraacetic acid
ESBL extended spectrum beta-lactamase

GFP green fluorescent protein
GIM-1 Germany imipenemase
HAI hospital-acquired infections
HIS Histidine
IAI intra-abdominal infection
ICARE intensive care antimicrobial resistance epidemiology
ICE Integration and conjugative element
ICU intensive care unit
IMP imipenemase
Inti integrase gene
IPTG Isopropyl-P-D-thiogalactoside
IS insertion sequence
ISCR insertion sequence common region
Kcat catalytic rate constant
KHM-1 Kyorin Health MBL
Km The Michaelis constant
KPC Klebsiella pneumoniae carbapenemase
LPS Lipopolysaccharide
MATE multi-drug and toxic compound extrusion family
MBL Metallo-p-lactamase
MDR multi-Drug Resistant
MFS major facilitator superfamily
xviii
MIC minimum inhibitory concentration
MIC 50 Minimum inhibitory concentration that kills 50% of the
bacteria
MIC 90 Minimum inhibitory concentration that kills 90% of the
bacteria
MDR Multi-drug resistance
MLST multilocus Sequence Typing
MYSTIC meropenem yearly susceptibility test information collection
NDM-1 New-Delhi metallo-p-lactamase
NI nosocomial infections
NP nosocomial pneumoniae
OM Outer membrane
OMP outer membrane protein
ORF open reading frame
OXA oxacillinases
PBP penicillin binding protein
PFGE pulsed field gel electrophoresis
qacAEl quaternary ammonium compound
RAPD Random amplified polymorphic DNA
RCS recombination crossover site
RNA ribonucleic acid
RND resistance nodulation division
xix
SHV sulfhydryl variable
SDS Sodium dodecyl sulphate
SIM-1 Seoul imipenemase
SLV single locus varian
SMR small multi-drug resistance
SPM-1 Sao Paolo metallo-p-lactamase
SSTI skin and soft tissue infection
sull sulphonamide resistance gene
SXT trimethoprim sulphamethoxazole
TEM Temoneira
TMB-1 Tripoli metallo-p-lactamase
tniC transposase gene
US united states
UTI urinary tract infection
VAP ventilator associated pneumonia
VIM Verona Imipenemase
XX
CONTENTS
Title page i
Declaration ii
Summary iii
Publications v
Acknowledgments vii
Dedication viii
List o f figures ix
List o f tables xvi
List o f abbreviations xvii
Chapter One
General Introduction
1.1 A ntibiotics................................................................ 2
1.1.1 Introduction................................................................ 2
1.1.2 History of antibiotics................................................... 2
1.2 Gram-negative bacteria........................................... 4
1.3 Examples of antibiotics used in treatment of
infections caused by bacteria................................. 10
1.3.1 plactam s....................................................................... 12
xxi
1.3.1.1 Cephalosporins............................................................ 12
1.3.1.1.1 Cefotaxim e................................................................. 12
1.3.1.1.2 Ceftazidim e................................................................. 13
1.3.1.1.3 Ceftriaxone................................................................. 13
1.3.1.2 Carbapenem s................................................................. 13
1.3.1.2.1 Im ipenem ..................................................................... 14
1.3.1.2.2 Meropenem ................................................................ 15
1.3.1.2.3 Ertapenem ..................................................................... 16
1.3.1.2.4 D oripenem ..................................................................... 17
1.4 Mode of antibiotic action ....................................... 17
1.4.1 Introduction................................................................. 17
1.4.1.1 Inhibition o f protein synthesis.................................. 18
1.4.1.2 Inhibition of cell wall synthesis.............................. 19
1.4.1.3 Inhibition of DNA synthesis....................................... 19
1.5 Mechanism of antibiotic resistance in Gram-
negative bacteria....................................................... 21
1.5.1 Efflux pump mediated antibiotic resistance 21
1.5.2 Outer membrane permeability and antibiotic
resistance.................................................................... 22
1.5.3 (3-lactamases................................................................. 24
1.5.3.1 Introduction................................................................. 24
1.5.3.2 Classification of p-lactam ases.................................. 25
xxii
1.5.3.3 Extended spectrum (3-lactamases (ESB L s) 25
1.5.3.4 Carbapenemases........................................................ 27
1.5.3.5 Class A carbapenemases ........................................... 28
1.5.3.6 Class D (3-lactamases............................................... 29
1.5.3.7 M etallo-p-lactamases............................................... 30
1.6 Global emergence of clinical antibiotic resistant
Gram-negative bacteria.......................................... 32
1.6.1 Evolution of antibiotic resistance in Gram-negative
bacteria...................................................................... 37
1.7 DNA structures that spread antibiotic resistance 44
1.7.1 Plasmids in multi-resistant Gram-negative bacteria 44
1.7.2 Pathogenicity islands (Multi resistance in bacteria) 45
1.7.3 Transposons................................................................ 47
1.7.4 Integrons..................................................................... 48
1.7.5 Insertion sequence common regions (ISC R s) 51
1.7.6 Insertion sequences................................................... 52
1.7.6.1 Integrative and conjugative elements (IC E ) 53
1.8 Objectives of stu d y .................................................... 55
Chapter Two
Methods and Materials 57
2.1 Bacterial collection................................................... 58
xxiii
2.. 1.1 Ethical considerations................................................ 58
2.2 Safety considerations 59
2.3 Bacterial strains used 59
2.4 Chemicals, reagents and radioactive lab els 59
2.5 Growth M edia............................................................ 60
2.5.1 Luria Bertani B ro th ................................................... 60
2.5.2 Luria Bertani A g a r................................................... 60
2.5.3 Mueller-Hinton A g a r............................................... 60
2.5.4 MacConkey Agar N o .3 ............................................... 61
2.5.5 MacConkey A g a r....................................................... 61
2.5.6 MacConkey Agar for isolation of ESBL/MBL
positive isolates........................................................... 61
2.5.7 S.O.C M edium ............................................................ 61
2.6 Sterilisation of M ed ia............................................... 61
2.7 Isolation of Environmental strains......................... 61
2.8 Etest experim ents........................................................ 62
2.9 Antimicrobial susceptibility testing and MIC
determ ination............................................................ 62
2.10 Phenotypic and genotypic detection of E S B L s 62
2.10.1 Amplification of DNA sequences using P C R 62
2.10.1.1 Amplification of blacrx-u type E S B L s..................... 62
2.10.1.2 Detection of blactx - m groupl and ISEcpl genes ... 65
xxiv
2.10.1.3 Amplification of 6/atem, blasuv, ampc, class 1
integrons and transposons..................................... 65
2.10.2 Phenotypic detection of M B L s................................ 66
2.11 Detection of blaoxA-48 and IS 1999 ......................... 66
2.12 Random amplified polymorphic DNA RAPD)
typing......................................................................... 67
2.12.1 RAPD DNA extraction by Chelex p re p .................. 67
2.12.2 Random amplified polymorphic DNA (RAPD-
P C R ).............................................................................. 68
2.12.3 DNA profile analysis by Agilent Bioanalzer 69
2.12.4 GelCompare analysis................................................... 69
2.13 Multilocus sequence ty p in g .................................... 69
2.14 Plasmid identification ............................................... 72
2.15 Transconjugation experim ents.................................. 73
2.16 Southern hybridization............................................... 74
2.16.1 Characterization of chromosomally and plasmid
mediated resistance genes ..................................... 74
2.16.1.1 Preparation of plugs of whole genomic D N A 74
2.16.2 Pulsed Field Gel Electrophoresis (PFG E )............. 76
2.16.3 Colony blotting............................................................ 77
2.16.4 In gel hybridization ................................................... 78
2.16.5 Labeling DNA p ro b es............................................... 79
XXV
2.17 Cloning experim ents.................................................... 80
2.18 Purification of TMB-1 ................................................ 81
2.18.1 Expression..................................................................... 81
2.18.2 Periplasm isolation.................................................... 82
2.18.3 Purification of the (3-lactamase................................... 82
2.18.4 G el-filtration................................................................. 83
2.19 Kinetic a ssa y ................................................................. 83
Chapter Three
3.1 Introduction................................................................. 85
3.2 R esu lts.......................................................................... 88
3.2.1 Antimicrobial sensitivity testing .............................. 88
3.2.2 Genotypic detection of E S B L S .................................. 89
3.2.2.1 The prevalence of CTX-M groups 1, 2, 8, 9 and 26 89
3.2.2.2 Detection of CTX-M-15 genes and I S E c p l 90
3.2.2.3 Detection of TEM and S H V ....................................... 94
3.2.2.4 CTX-M groupl type E S B L s....................................... 97
3.2.2.5 Detection of blaoxA-4 » and IS1999 99
3.2.3 Characterisation of plasmids carrying blacix-M
groupl and I S E c p l.................................................. 99
3.2.4 Typing o f K. pneumoniae by R A P D .......................... 102
3.2.5 Molecular typing o f K. pneum oniae.......................... 105
xxvi
3.2.6 Multilocus sequence ty p in g ....................................... 110
3.2.7 Detection of chromosomally / plasmid mediated
blacxx-u groupl........................................................ 112
3.2.8 Transconjugation experim ents................................... 113
3.2.9 Detection of plasmid mediated blacix-u group 1 in
parents and transconjugants..................................... 117
3.2.10 Detection of the movement of ISEcpl from parents
to transconjugants.................................................... 117
3.2.11 Plasmid T yping............................................................ 122
3.2.12 Detection of mobile genetic elem ents...................... 122
3.2.12.1 Class 1 integrons........................................................ 122
3.2.12.2 Identification of transposons....................................... 125
3.2.12.3 Transposase encoding g en es....................................... 127
3.2.12.3.1 PFGE o f SI genomic digests and probing with tniC 127
3.2.12.4 Detection of ISCR elem ents....................................... 130
3.3 D iscussion..................................................................... 132
Chapter Four
4.1 Introduction................................................................. 139
4.2 R esu lts......................................................................... 141
4.2.1 Characterisation of E. coli isolates and
antimicrobial susceptibility testing........................ 141
xxvii
4.2.2 Detection of TEM, SHV and CTX-M type ESBLs 141
4.2.3 Transconjugation experim ents.......................... 142
4.2.3.1 Antibiotic resistance profile of E. coli
transconjugants................................................... 143
4.2.4 Plasmid typing of ESBL positive E. c o li......... 148
4.2.5 Detection of plasmid mediated blacix-M groupl and
ISEcpl in parents and transconjugants of E. c o li... 149
4.2.6 Typing of E. coli isolates................................... 154
4.2.7 Detection of chromosomally mediated blacix-u
groupl ........................................................................ 158
4.2.8 Detection of class 1 integrons and Tn402
transposons.......................................................... 161
4.3 D iscussion.......................................................... 162
Chapter Five
5.1 Introduction................................................................ 168
5.2 R esu lts................................................................. 170
5.2.1 Antibiotic susceptibility testin g .................................. 170
5.2.2 Detection of MBLs using E te st.................................. 170
5.2.3 Detection of MBL encoding g en es......................... 170
5.2.4 Transconjugation experim ent........................... 173
5.2.5 Typing of P. aeruginosa............................................... 174
xxviii
5.2.6 Detection of chromosomally and plasmid mediated
blay\u-i....................................................................... 174
5.2.6.1 Characterisation of chromosomal /plasmid
mediated blay\u - 2 ..................................................... 174
5.2.7 Detection of class 1 integrons and transposons 180
5.3 D iscussion..................................................................... 183
Chapter Six
6.1 Introduction................................................................. 188
6.2 R esu lts......................................................................... 190
6.2.1 Analysis o f samples from Tripoli hospitals 190
6.2.2 Genetic analysis of carbapenem resistance A.
xylosoxidans strains AES301 ................................. 190
6.2.3 Cloning and transconjugation experim ents 192
6.2.4 Genomic location of &/<ztmb-i ..................................... 193
6.2.5 Comparison of TMB-1 with other MBLs ............. 193
6.2.6 Kinetic properties of TMB-1 ...................................... 199
6.3 D iscussion..................................................................... 201
Chapter Seven
General Discussion................................................... 205
xxix
Chapter Eight
Appendices 216
Appendix A 217
Table A. 1 Antibiotics and chemicals used in experiments ... 217
Table A.2 Oligonucleotides used for PCR experim ents 218
Appendix B 221
Figure B.l Multiplex PCR to detect CTX-M- groups 1, 2, 8, 9
and 26 ......................................................................... 221
Figure B.2 Multiplex PCR to detect CTX-M- groups 1, 2, 8, 9 221
and 2 6 ..........................................................................
and 2 6 ........................................................................
and 2 6 .........................................................................
Fig. B.5 Multiplex PCR to detect CTX-M- groups 1, 2, 8, 9 223
and 2 6 ........................................................................
Fig. B.6 PFGE o f SI digests of K. pneum oniae................... 224
Fig. B.7 Autorad of after probing o f PFGE gel from fig.B.6 225
Fig. B.8 DNA sequence amplified from K. pneumoniae
XXX
A E S 5 9 .......................................................................................... 226
Fig. B.9 Alignment of DNA from fig. B .8 ............................ 226
AES59 ......................................................................... 230
Fig. B.l 1 Alignment of DNA from fig. B .1 0 .......................... 230
Fig. B .l2 DNA sequence amplified from K. pneumoniae
AES 8 .......................................................................... 232
Fig. B.l 3 Alignment of DNA from fig. B.l 2 .......................... 232
AES48 ........................................................................ 234
Fig. B.l 5 Alignment of DNA from fig. B .1 4 .......................... 234
AES48 ...................................................................... 237
Fig B.l 7 Alignment of DNA from fig. B.l 6 .......................... 237
AES74 ...................................................................... 240
Fig. B.l 9 Alignment of DNA from fig. B.l 8 .......................... 240
AES 140 .................................................................... 242
Fig. B.21 Alignment of DNA from fig. B.20 .......................... 242
AES 140 .................................................................. 244
xxxi
Fig. B.24 DNA sequence amplified from K. pneumoniae 246
AES261 .....................................................................
A E S 817 ..................................................................... 247
AES984 .................................................................... 249
AES 1001 .................................................................. 252
Fig. B.32 Alignment of RpoB gene from K. pneumoniae
AES817 ................................................................... 255
Fig. B. 33 Alignment of Gap A gene from K. pneumoniae
A E S 817................................................................... 255
Fig. B.34 Alignment of infB gene from K. pneumoniae
A E S 817.................................................................. 256
Fig. B.35 Alignment of Pgi gene from K. pneumoniae
A E S 817..................................................................... 256
Fig. B.36 Alignment of PhoE gene from K. pneumoniae
xxxii
A E S 8 1 7 .................................................................................. 257
Fig. B.37 Alignment of tnoB gene from K. pneumoniae
A E S 817..................................................................... 257
Fig. B.38 Alignment of mdh gene from K. pneumoniae
A E S 817..................................................................... 258
Fig. B.39 Alignment of mdh gene from K. pneumoniae
AES809 ..................................................................... 258
Fig. B.40 Alignment of Pgi gene from K. pneumoniae
AES809 ...................................................................... 259
Fig. B.41 Alignment of GapA gene from K. pneumoniae
AES809 ..................................................................... 259
Fig. B.42 Alignment o f PhoE gene from K. pneumoniae
AES809 ........................................................................ 260
Fig. B.43 Alignment of tnoB gene from K. pneumoniae
AES809 ..................................................................... 260
Fig. B.44 Alignment of infB gene from K. pneumoniae
AES809 ...................................................................... 261
Fig. B.45 Alignment of RpoB gene from K. pneumoniae
AES809 ...................................................................... 261
Fig. B46 Alignment of Pgi gene from K. pneumoniae
AES808 .................................................................. 262
Fig. B47 Alignment of tnoB gene from K. pneumoniae
xxxiii
A E S 8 0 8 .................................................................................... 262
Fig. B48 Alignment of PhoE gene from K. pneumoniae
AES808 ....................................................................... 263
Fig. B49 Alignment of infB gene from K. pneumoniae
AES808 ..................................................................... 263
Fig. B50 Alignment of RpoB gene from K. pneumoniae
AES808 ................................................................... 264
Fig. B51 Alignment of mdh gene from K. pneumoniae
AES808 ..................................................................... 264
Fig. B52 Full sequence of AES81 integron 265
Fig. B53 Full sequence of AES83integron 266
Fig. B54 Full sequence of AES 135 integron 267
Appendix C 268
Table C.l List o f E. coli isolates used in experim ents 268
Fig. C.l DNA sequence from E. coli AES226 ...................... 269
Fig. C.2 Alignment of DNA from fig. C.l .............................. 270
Fig. C.3 DNA sequence from E. coli AES228 ...................... 274
Fig. C.4 Alignment of DNA from fig. C .3 ............................ 274
Fig. C.5 DNA sequence from E. coli AES232 ...................... 277
Fig. C.6 Alignment of DNA from fig. C .5 ............................ 277
Fig. C .l DNA sequence from E. coli AES228 ...................... 280
Fig. C.8 Alignment of DNA from fig. C . l ........................... 281
xxxiv
Fig. C.9 DNA sequence from E. coli AES226 ..................... 282
Fig. C.10 Alignment of DNA from fig. C .9 ........................... 283
Fig. C.l 1 DNA sequence from E. coli AES232 ..................... 285
Fig. C.l 2 Alignment of DNA from fig. C.l 1 ........................... 285
Appendix D 287
Table D .l Environmental isolates collected from T ripoli 287
Fig. D. 1 Hydrolysis of antibiotic meropenem by TMB-1 ... 289
Fig. D.2 Hydrolysis of antibiotic ertapenem by TMB-1 ......... 290
Fig. D.3 Hydrolysis of antibiotic ceftazidime by TMB-1 ... 291
Fig. D.4 Hydrolysis of antibiotic ampicillin by TMB-1 ........ 292
Fig. D.5 Hydrolysis of antibiotic imipenem by TMB-1 ........ 293
Fig. D.6 Hydrolysis of antibiotic cefoxitin by TMB-1 ........ 294
Fig. D.7 Hydrolysis of antibiotic cefuroxime by TMB-1 ... 295
Fig. D.8 Hydrolysis of antibiotic piperacillin by TMB-1 ... 296
Fig. D.9 Full sequence of 3kb integron from A. xylosoxidans 297
Chapter Nine
Bibliography 300
XXXV
Chapter One
General Introduction
1.1 Antibiotics
1.1.1 Introduction
Selman Waksman was one of the most recognized investigators in the field of
bacteriology in 1940’s, Waksman defined the term “antibiotic” as the
substance that has the ability to kill bacteria (Bush, 2010a; Waksman &
Woodruff, 1942). The term was singularly used to refer to a molecule that was
bacteriostatic or bactericidal; however, today, the definition has changed and
expanded - it is applied to natural products and synthetic chemicals that have
antibacterial and antifungal activities. (Bush, 2010).
1.1.2 History of antibiotics

Antibiotics were introduced in the 1930’s as a result of the discovery of the
antibiotic penicillin from the fungus Penicillium notatum by Alexander
Fleming in 1928 and the prontosil (sulfonamidochrysoidine) discovered by
Gerhard Domagk in 1932. Such discoveries had a profound impact on human
health and provided rapid and effective treatment of patients suffering from
bacterial infections known to have been fatal. (Butler & Cooper, 2011).
P-lactam antibiotics were introduced clinically in 1940s exemplified by the
antibiotic penicillin to treat bacterial infections caused by human pathogenic
bacteria after approval of Food and Drug Administration (FDA) as before this
time o f the antibiotic era, infections such as bacteraemia caused by
Streptococcus pneumoniae were the causative agents of mortality (Coates et
al., 2002; Dineen et al., 1976). The introduction of antimicrobial agents helped
2
to decrease the mortality rates, e.g. the subcutaneous use of sulfanilamide
caused reduction o f acute meningococcal meningitis from 70-90% to nearly
10% (Powers, 2004). Between the 1930s and 1960s, more than 20 new classes
o f antibiotics were discovered - mainly natural or semi-synthetic (Table 1.1).
As a result o f these antibiotics to treat severe and life-threatening infections,
the story has become a successful one (Butler & Buss, 2006; Powers, 2004)
and has led to an over confidence on the ability of antimicrobials to eradicate
all infectious diseases.
After the 1960s, research for new and novel drugs slowed and pharmaceutical
industry paid less attention to antimicrobial research (Boucher et al., 2009).
This in part can be explained by the difficulty in discovering new antibacterial
agents with completely novel mechanisms of action and also the cost of
research - particularly clinical trials. (Coates et al., 2002; Powers, 2004).
Since the intensive work on antimicrobial agents in the 20th century, only two
new classes of antibiotics; daptomycin (Figure 1.1) and oxazolidinones
(Figure 1.2) have recently been utilised to treat Gram-positive infections,
whereas, innovation to address Gram-negative bacteria is still struggling and,
at best, can only rely on modification of existing drugs e.g. fluoroquinolones
(Figure 1.3), aminoglycosides (Figure 1.4), tetracyclines (Figure 1.5) and 13-
lactams. (Figure 1.6) (Bush & Pucci, 2011).
3
1.2 Gram-Negative Bacteria
Gram-negative bacteria are micro-organisms that are known to have an outer
“cell envelope” or outer membrane (OM), which differs considerably from
other bacterial strains in terms of structure and function. This “cell envelope”
is composed o f three envelope layers; the OM layer, the periplasm and the
inner membrane or cytoplasmic membrane (Figure 1.7) (Gupta, 2011).
The structure o f the OM has a unique lipid bilayer and its layers of
phospholipids are confined to the inner side of the OM ( Silhavy et al., 2010).
Glycolipids are main components of the OM and they are principally
lipopolysacharides (LPS) which are located as an outer leaflet of the Gram-
negative OM and play an important role as a functional barrier. LPS comprises
the core o f polysaccharide, lipid A, and extended polysaccharide chain O
antigen. Lipid A is also known as the endotoxin, minute amounts of which can
cause fever and septic shock syndrome. (Ryan et al., 2004; Silhavy et al.,
2010 ).
The OM contains proteins which differ to proteins of the cytoplasmic
membrane. Those proteins are classified into two groups; lipoproteins and 0-
barrel proteins. The function of most lipoproteins are not known yet, whereas
the p-barrel proteins are known as Outer Membrane Proteins (OMPs) and
have different roles according to the kind of OMP, for instance the function of
4
OmpF and OmpC are known as porins in E. coli allows the passive diffusion
and facilitated movement of monosaccharides, disaccharides and amino
Table 1.1 History of antibiotic introductions and approval (according to

Powers, 2004)
Antibiotic Year o f Discovery
Sulfonamides 1935 (launched)
B-lactams 1941 (launched)
Aminoglycosides 1944 (introduced)
Streptomycin 1947 ( launched)
Chloramphenicol 1949 (launched)
Tetracycline 1950 ( launched)
Macrolides 1952 ( introduced )
Glycopeptides 1956 ( introduced)
Rifamycins 1957 (introduced)
Nitromidiazoles 1959 (introduced)
Quinolones 1962 (introduced)
Nalidixic acid 1964 (introduced)
Gentamicin 1967 (launched)
Trimethoprim 1968 (launched)
Oxazolidinones 2000 (launched)
Linezolid 2000 (launched)
Lipopeptides 2003 (launched)

... ...... ..................... .
5
HaC—(CHj )a— C —Trp—Asr—Asp—Thr— Gly—Om—Asp—D— Ala—Asp—Gly—D—Ser—
COOH
Figure 1.1 Chemical structure of Daptomycin (according to Bush &

Pucci, 2011)
Figure 1.2 Chemical structure of the oxazolidinone radezolid (according

to Bush & Pucci, 2011)
O O
HO
Figure 1.3 Chemical structure of the fluoroquinolone delafloxacin

(according to Bush & Pucci, 2011)
6
MH
OH
Figure 1.4 Chemical structure of the aminoglycoside ACHN-490

OH O OH O
Figure 1.5 Chemical structure of the tetracycline omadacycline

O
h 2n
J^
o
J N^ i
N
bso3H
Figure 1.6 Chemical structure of the Avibactam NXL-104 (according to

Bush & Pucci, 2011)
7
Gram-positive Ce/tWaff
►Peptidoglycan
Protein
n®n n®n nfl n n®nn nniin nnnn \ cytoplasmic

Eh°-^piûuUUuUUOUUUUUUUWUUUOy J M em brane
Cytoplasm
Gram-negative Cell Wa/f
Protoin [1 LPSt lj [1 Porir
Mn$nnfinn#nH!in nnnnn pouter

ûuuuluuouuuuussuw uuui M em b ran e
P er ip la sm ic
} Space
nnnnnnnsnnnnnnnnnnnon
P ep tid o a lv c a n t
p h c s p j ^ p j i^ j iy y ^ y m j y y ^ y y y g ^ y y y y y
Inner
} M em b ran e
Cytoplasm
Figure 1.7 Cell wall envelopes of Gram-positive and Gram-negative

bacteria (LPS: Lipopolysaccharide; LTA: Lipoteichoic acid)
(http://www.cehs.siu.edu/fix/medmicro/genmicr.htm)
8
acids across the OM (Sihavy et al., 2010; Greenwood, 2007; Ryan et al.,
2004). The OMPs in Klebsiella pneumoniae; OMPK35 and OMPA36, act as a
channel for antibiotics to pass through these porins to the cytoplasm and losing
either has shown to facilitate resistance to cephalosporins (Tsai, et al., 2011 ).
The periplasm lies between the two membranes (Figure 1.7) and is filled with
a fluid called the periplasmic gel and situated between the outer and the inner
membranes and considered as the interior part of the cell envelope. The
periplasm plays a crucial role as a transporter of sugars and amino acids and
because it is densely packed with proteins, it acts to sequester of the harmful
RNAse and alkaline phosphatase degradative enzymes. The periplasm is
inhabited with periplasm binding proteins and chaperon like molecules, both
have different functions. Periplasm binding proteins act as transporter of
sugars and amino acids as well as chemotaxis, whereas chaperon like
molecules function in envelope biogenesis (Silhavy et al., 2010) such as the
movement o f synthesised molecules e.g. LPS from the cytoplasm to across
the periplasm be assembled on the outer membrane, specific transporters are
required; the periplasmic protein LptA, the OM lipoprotein LptE and the p-
barrel OM protein LptD. (Ruiz et al., 2009). Chromosomal, plasmid-mediated
or inducible p-lactamases present in the periplasm play an important role in
protecting the PBPs from p-lactam antibiotics (Sykes & Matthew, 1976).
9
The cell wall consists of a thin layer of peptidoglycan known as murein 5-10
(nm) linked to the outer membrane via lipoproteins. N-acetylglucosamine and
N-acetylmuramic acid molecules represent the main structure o f the
peptidoglycan layer; moreover, they are cross-linked with penta-peptide side
chains (Vollmer et al., 2008). Despite the fact that the peptidoglycan in Gram-
negative bacterial cell wall is greatly reduced, it plays a significant role in
giving the cell its stability and rigidity and, accordingly, determines cell shape.
The reason for this is the composition o f glycan chains in the form of N-
acetylglucosamine-N-acetylmuramic acid, which is found linked in alternative
ways to form murein saculus heteropolymer. The penicillin binding proteins
(PBPs) play a major role in the polymerization of the glycan strand that is
called transglycosylation. PBPs are the target of p-lactam antibiotics but are
protected by p-lactamases in the periplasm (Sauvage et al., 2007).
1.3 Examples of antibiotics used in treatment of infection caused by
bacteria
Gram-negative bacteria are a leading cause of life-threatening infections and
include nosocomial infections (NI), nosocomial pneumonia (NP), urinary tract
infections (UTIs), intra-abdominal infections (IAIs), pediatric bacterial
meningitis, septicaemia, neutropenia, community acquired infections (CAIs),
and pelvic inflammatory diseases (Lamb et al., 2002; Plosker et al., 1998;
Chaudhuri et al., 2011; Baughman, 2009). Since the discovery of antibiotics,
many classes o f antibiotics have been employed and derivatives of established
10
antibiotics trialed to overcome increasing resistance. (Table 1.2) (Coates et al.,
2002 ).
Table 1.2 Main classes and examples of antibiotics and P-lactamase

inhibitors (according to Coates et a l 2002)____________________________
Class Examples
P-lactams
Penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin,
Penicillins ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin,
temocillin
Cephalosporins
First generation Cepalothin, cephapirin, cephradine, cephaloridine, cefazolin
Cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor, loracarbef, cefoxitin,
Second generation
cefmetazole
Cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime,
Third generation
cefpodoxime, ceftibuten, cefdinir
Fourth generation Cefpirome, cefepime
Carbapenems Imipenem, meropenem
Monobactams Aztreonam
fi-lactamase inhibitors Clavulanate, sulbactam, tazobactam
Streptomycin, neomycin, kanamycin, paromycin, gentamicin, tobramycin, amikacin,
Aminoglycosides
netilmicin, spectinomycin, sisomicin, dibekacin, isepamicin
Tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline,
Tetracyclines
methacycline, doxycycline
Rifampicin (also called rifampin), rifapentine, rifabutin, bezoxazinorifamycin,
Rifamycins
rifaximin
Macrolides Erythromycin, azithromycin, clarithromycin
Lincosamides Lincomycin, clindamycin
Glycopeptides Vancomycin, teicoplanin
Streptogramins Quinupristin, daflopristin
Sulphanilamide, /wra-aminobenzoic acid, sulfadiazine, sulfisoxazole,
Sulphonamides
sulfamethoxazole, sulfathalidine
Oxazolidinones Linezolid
Nalidixic acid, oxolinic acid, norfloxacin, pefloxacin, enoxacin,
ofloxacin/levofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin,
Quinolones
grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin,
sitafloxacin
Others Metronidazole, polymyxin, trimethoprim
11
1.3.1 p-lactams
1.3.1.1 Cephalosporins
Cephalosporins are class of antimicrobials used to treat bacterial infections
due to Gram-negative and Gram-positive bacteria. Cephalosporins are divided
to 1st, 2nd, 3rd, 4th and 5th generations. The 1st generation was first introduced
in 1945 as natural product derivatives to disrupt the cell wall by interrupting
the synthesis o f peptidoglycan causing lysis of bacteria. (Butler & Buss,
2006). Third generation cephalosporins are among the most widely used
subclass of antibiotics and include cefotaxime, ceftazidime, and ceftriaxone.
This class of antibiotics is administered to treat hospital acquired infections
particularly to eradicate infections caused by Enterobacteriaceae e.g. K.
pneumoniae and Escherichia coli.
1.3.1.1.1 Cefotaxime
Cefotaxime has a broad-spectrum o f activity and plays an important role in the
treatment o f Gram-negative bacterial infections in adult and pediatric patients.
It is administrated to treat bacterial infections due to skin and soft tissue
infections, nosocomial infections, pneumonia, complicated urinary tract
infections, meningitis, bone and joint infections and bacteraemia (Adu &
Armour, 1995; Plosker et al., 1998; Dajani, 1995).
12
1.3.1.1.2 Ceftazidime
Ceftazidime is an aminothiazolyl syn-methoxyimino cephalosporin, it is a p-
lactam antibiotic has broad-spectrum activity against Gram-negative.
Ceftazidime is administered to treat bacterial infections e.g. respiratory tract,
genitourinary tract, gynecological, bone and joint, septicaemia, intra
abdominal, bacteraemia, meningitis, skin and tissue and ventilator associated
pneumoniae infections (VAPs). (Buijk et al., 2002; Lorente et al., 2007).
1.3.1.1.3 Ceftriaxone
Ceftriaxone was introduced in 1980s and used extensively to treat bacterial
infections due to its stability against P-lactamases, particularly produced by
members of Enterobacteriaceae. It is used to treat broad range of infections;
these include meningitis in adults and infants, acute otitis media, CAIs,
uncomplicated gonorrhea, pelvic inflammatory disease, acute pyelonephritis
and spontaneous bacterial peritonitis. (Lamb, et al., 2002; Jones, et al., 1998;
Diekema, et al., 1999).
1.3.1.2 Carbapenems
Carbapenems are derived from the antibiotic thienamycin which is a natural
product o f the Gram-positive bacterium Streptomyces cattleya. This class of p-
lactams includes meropenem, imipenem, ertapenem, and doripenem.
Carbapenems are often used as empirical therapy and to treat bacterial
infections caused by Gram-negative bacteria that produce resistant
13
determinants against extended spectrum cephalosporins. Carbapenems are
classified into two groups. Group 1 comprises antibiotics that have limited
antibacterial activity against non-fermenters Gram-negative bacteria such as
ertapenem. Group 2 includes antibiotics active against non-fermenters and
recommended to treat nosocomial infections. (Shah & Isaacs, 2003; Livermore
and Woodford, 2000; Bimbaum et al., 1985; Ayalew et al.,2003; Zhanel et al.,
2007; Mohr, 2008).
1.3.1.2.1 Imipenem
Imipenem is A-formimodoyl-thienamycin (Figure 1.8) is not used on its own
because it is rapidly degraded by dehydropeptidase produced by the human
kidney and has an adverse toxic effect on the kidney, therefore imipenem
should be co-administrated with cilastatin in the ratio of 1:1 to act as an
inhibitor o f the dehydropeptidase enzyme and to neutralize the toxic effect of
the antibiotic. (Rodloff et al., 2006).
Transpeptidases also known as penicillin binding proteins (PBPs) cross link
the peptidoglycan and provide the bacteria with a rigid cell wall are the main
targets for imipenem. Imipenem has been shown to inactivate the
transpeptidase of PBP-1A, PBP-1B and PBP-2, it moreover, inhibits the D-
alanine carboxypeptidase o f PBP-4 and PBP-5 in E. coli. (Hashizume et al.,
1984). Imipenem is a broad-spectrum antibiotic indicated as initial empirical
therapy and in treating serious bacterial infections including NI, ventilator
14
associated pneumonia (VAP), febrile neutropenia (Torres et al., 2000; Zanetti
et al., 2003; West et al., 2003; Raad et al., 2003; Cherif et al., 2004), hospital
acquired pneumonia (HAP), healthcare associated pneumonia (HCAP),
patients hospitalized suffering from intra-abdominal infections, patients with
skin and soft tissue infections and lower respiratory tract infections (Neu,
1983; Shah & Isaacs, 2003).
1H
OH H
NHCH
H3C H-.0
COOH
Figure 1.8 (A-formimodoyl-thienamycin). (Rodloff et al., 2006).
1.3.1.2.2 Meropenem
Meropenem is a member o f carbapenems marketed to eradicate Gram-
negative bacterial infections and was approved by the FDA in 1996 (Zhanel et
al., 2007; Baldwin et al., 2008). Meropenem binds effectively to penicillin
binding protein (PBP) with high affinity, accordingly inhibiting the growth of
the micro-organism. It has high affinity to PBPs 2, 3, and 4 of E. coli and
PBPs 1 and 2 of Pseudomonas aeruginosa (Baldwin et al., 2008).
15
Meropenem is effective in the treatment of several infectious diseases caused
by pathogenic bacteria, it is recommended for the treatment of NP, it can also
be used as an alternative to other antibiotics such as amikacin (Alvarez Lerma,
2001) or combinations of antibiotics e.g. ceftazidime and tobramycin
(Heyland et al., 2008). Meropenem is also very efficacious in treating patients
with complicated intra-abdominal infections (CIAI) (Zanetti et al., 1999;
Brismar et al., 1995). In one study, 153 patients with septicaemia, meropenem
was effective as an empirical therapy, and as effective as ceftazidime with or
without amikacin (Baldwin et al., 2008). Meropenem also displays high
efficacy in treating adults and paediatric patients suffering from cancer related
febrile neutropenia infected with E. coli, Klebsiella spp and P. aeruginosa
(Oguz et al., 2006; Kutluk et al., 2004; Feld et al., 2000; Cometta et al., 1996),
and patients with bacterial meningitis caused by K. pneumoniae and
Haemophilus influenzae (Odio et al., 1999; Schmutzhard et al., 1995). It is
also highly active in treating complicated urinary tract infections (CUTI) (Cox
et al., 1995); complicated skin and skin structure infections (CSSSIs) (Fabian
et al., 2005) and acute pulmonary infections caused by P. aeruginosa in
patients with cystic fibrosis (Blumer et al., 2005).
1.3.1.2.3 Ertapenem
Ertapenem has a broad-spectrum activity against Gram-negative bacteria but
not non-fermenters as it has limited antibacterial activity and it is
recommended for CAIs (Keating & Perry, 2005). Ertapenem is active against
16
Enterobacteriaceae producing extended-spectrum p-lactamases (ESBLs) and
AmpC P-lactamases. Ertapenem binds to PBPs, subsequently interferes with
bacterial cell wall synthesis and due to occurrence o f lp-methyl substituent,
co-administration with cilastatin with ertapenem is not required as ertapenem
is stable against renal dehydropeptidase I. (Alhambra et al., 2004)..
1.3.1.2.4 Doripenem
Doripenem was approved by FDA in 2007 to be used to treat CIAI and CUTIs
(Paterson & Daryl, DePestel, 2009). Its activity resembles that of meropenem
(Jones et al., 2005a; Mushtaq et al., 2004). Doripenem forms a stable acyl-
enzymes and causing weakness bacterial cell wall and consequently lead to
cell wall rupture as a result of osmotic pressure forces (Stratton, 2005). PBP2
and PBP3 in P. aeruginosa and E. coli are the prime targets for doripenem
(Davies et al., 2008).
Doripenem is very similar to meropenem in the treatment of post-surgical
infections (Lucasti et al., 2008); and can be employed to treat CUTIs,
pyelonephritis and baseline bacteremia, hospital acquired pneumonia
including VAP (Rea-Neto et al., 2008; Chastre, et al., 2008).
1.4 Mechanism of antibiotic action
1.4.1 Introduction
Antibiotics were discovered and introduced as to be used to treat bacterial
infections by interrupting the physiological mechanisms inside the bacterial
17
envelope/cytoplasm that allow normal cellular function. Two main
mechanisms o f bacterial inhibition are known, bactericidal drugs induce cell
death while bacteriostatic drugs act as cell growth inhibitors (Kohanski, et al.,
2010). Herein, I will describe the effect of antibiotics on protein synthesis, cell
wall and DNA synthesis.
1.4.1.1 Inhibition of protein synthesis
Protein synthesis occurs at the ribosome of bacteria and during phases of
synthesis, initiation, elongation and termination, more specifically on the 50S
and 30S subunits (Figure 1.9). Inhibitors of protein synthesis differ according
to the target site, inhibitors of 5OS subunit of Gram-negative bacteria include
lincosamide e.g clindamycin and chloramphenicol (Katz & Ashley, 2005).
Aminocyclitol family and tetracyclines are among the 30S ribosome inhibitors
and include kanamycin, gentamicin and streptomycin. These antibiotics inhibit
the bacterial growth by interrupting the access of aminoacyl-tRNAs to the
ribosome (Chopra & Roberts, 2001). Protein mistranslation can also occur as
a result o f the interaction between aminoglycosides and 16S rRNA, such
interaction causes alteration in the complex between mRNA and aminoacyl-
tRNA at the ribosome and consequently mismatching of tRNA will take place
leading to protein mistranslation (Pape et al., 2000).
18
Aminoglycosides
Aminoglycoside
ip iifflr a Outer
mRNA m m m m m membrane
Inner
Mbosome
protein membrane
Increased
^ 7 —-v v aminoglycoside
uptake
Figure 1.9 Inhibition of protein synthesis by aminoglycosides (according

to Kohanski et al., 2010)
1.4.1.2 Cell wall synthesis
The bacterial envelope is enclosed by a covalently cross-linkage of
peptidoglycan layers, these layers are composed of peptide P-(l-4)-V-acetyl
hexosamine. (Bugg & Walsh, 1992). The integrity of the bacterial cell wall is
likely to be affected by the degree of peptidoglycan cross-linking (Holtje,
1998). As mentioned previously, p-lactams are the largest group of antibiotics
that target the cell wall (Figure 1.10). Glycopeptides also share this target.
Carbapenems and cephalosporins are important classes of antibiotics used as a
therapy, their mechanism of action is represented in blocking the cross-linking
of peptidoglycan units, such blocking is achieved by the inhibition of PBP by
means of transpeptidase. (Kohanski et al., 2010).
1.4.1.3 Inhibition of DNA synthesis

Quinolone antibiotics are DNA synthesis inhibitors that act by targeting DNA
gyrase that is known as topoisomerase II and topoisomerase IV which is
19
known as topoIV (Figure 1.11). These antibiotics prevent the rejoining of the
DNA strand at the DNA cleavage stage and consequently affect the synthesis
of DNA and cause cell death. It has been shown that quinolone antibiotics
target topoisomerase II in Gram-negative bacteria e.g. E. coli and Neisseria
gonorrhoeae (Drlica et al., 1978; Kohanski et al., 2010).
p-lactams
G ra m n e g a tiv e
Outer
membrane
P-lactam
Lysis and
cell death
Autolysin
Inner
membrane
Figure 1.10 Inhibition of cell wall synthesis by P-Iactams (according to

Kohanski et al., 2010)
Quinolones
DNA polymerase Protein independent

complex
x m g g x x x x
Quinolones
/
SOS
\
►DNA repair
\ /
Topoisomerase
x x x x x x x x
Protein dependent
Figure 1.11 Inhibition of DNA synthesis by quinolones (according to

Kohanski et al., 2010)
1.5 Mechanism of antibiotic resistance in Gram-negative bacteria
Several factors have been attributed to the ascending level of bacterial
resistance to antimicrobial agents used in clinical settings and have led to the
emergence of multi-drug resistant strains.
1.5.1 Efflux pump mediated antibiotic resistance
Efflux is considered one major mechanism by which bacteria can expel
antimicrobials outside the cell. Efflux pumps are often chromosomally
mediated; however, some plasmid mediated pumps have been reported. Five
families of efflux pumps were reported, ATP binding cassette superfamily
(ABC), the multi-drug and toxic compound extrusion family (MATE), the
major facilitator superfamily (MFS), the small multi-drug resistance family
(SMR) and the resistance nodulation division superfamily (RND). (Li &
Nikaido, 2004; Li & Nikaido, 2009).
Single or multi-drug resistance in E. coli is in part attributed to the occurrence
of efflux transports in addition to other resistance mechanisms. More than 37
efflux pumps were found in the genome of E. coli belonging to different
families; seven RND type, seven ABC type, 1 MATE type and 19 MFS.
AcrAB is known to work with the outer membrane protein TolC as the
combination system shows broad substrate specificity toward (3-lactams,
chloramphenicol and novobiocin as well as dyes, detergents and organic
solvents (Li & Nikaido, 2004). Twelve types of RND type efflux system have
21
been described as responsible for resistance of P. aeruginosa to
antimicrobials, detergents, chemicals, molecules, dyes and antiseptics for
instance MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexGHI-OprD and
MexXY efflux pumps. MexAB-OprM efflux provides a wide range of
resistance to antibiotics, p-lactam, tetracycline, trimethoprim, chloramphenicol
with intrinsic resistance toward flouroquinolones (Askoura et al., 2011).
1.5.2 Outer membrane permeability and antibiotic resistance
The outer membrane in Gram-negative bacteria has already been described in
(section 1.2). Antibiotics undertake two pathways to penetrate the outer
membrane targeting the cytoplasmic membrane; the lipid-mediated pathway
and general porin diffusion. Some antibiotics use both ways to enter the cell
e.g. tetracycline and quinolones. Hydrophobic antibiotics enter the Gram-
negative bacterial outer membrane via the lipid-mediated pathway whereas the
hydrophilic antibiotics use porins to reach their target (Delcour, 2009).
Gentamicin, kanamycin, erythromycin, rifamycin, fusidic acid and cationic
peptides are known as hydrophobic antibiotics able to enter the cell through
the outer membrane bilayer (Vaara, 1992; Nikaido, 2003).
Bacteria use the LPS core region as a barrier for hydrophobic antibiotics.
Some antibiotics and chemicals play a major role in the sensitivity of bacteria
to antimicrobials, e.g. Tris/EDTA and polymyxin B. The target of Polymyxin
B is the cytoplasmic membrane; it penetrates the cell and by binding to
negatively charged LPS causes destabilisation of the outer membrane, the fatty
22
acid tail o f the antibiotic causes disruption to the membrane integrity leading
to the antibacterial action. Resistance of bacteria to polymyxin B is achieved
by esterification of the lipid A phosphates by the occurrence of 4 to 6 times of
4-aminoarabinose and more phosphoethanolamine, these compounds lower the
negative charge of the LPS leading to more resistance to polymyxin B
penetration (Cardoso, 2007; Delcour, 2009).
The term porin refers to p barrel proteins that act as a channel crossing the cell
membrane. The classical porins that are known to facilitate the diffusion of
molecules are OmpC and OmpC subfamilies; however, some exceptions
should be taken into consideration such as PhoE in E. coli and OprD of P.
aeruginosa and others, (http://www.membranetransport.org/). The porin
channel provides an entry for p-lactams and fluoroquinolones but Gram-
negative bacteria have developed some mechanisms to withstand antibiotics,
such as changing porin type or the levels expressed, modification of the target
site and synthesis o f pore blocking molecules. (Pages et al., 2008). For
example, OmpK35, one of the characteristic porins of K. pneumoniae and of
the OmpF porin group was replaced with OmpK36 as a result of the exposure
to treatment of patients harbouring the K. pneumoniae with P-lactam
antibiotics (Domenech-Sanchez et al., 2003). In vivo and in vitro evidence
show that mutation occurred in OprD of P. aeruginosa causing carbapenem
resistance in the presence or absence of carbapenemase production (Ochs et
al., 2000; Wolter et al., 2004).
23
1.5.3 p-lactamases
1.5.3.1 Introduction
The term p-lactamase refers to the enzymes produced by micro-organisms that
hydrolyses p-lactam molecules and thus singularly or in part enables p-lactam
resistance. More than 500 p-lactamase enzymes have been reported to date
(www.lahey.org/studies.webt.htm). It is considered the most common P-
lactam resistance mechanisms that contribute to wide spread resistance among
Gram-negative bacteria (Bush & Jacoby, 2010). p-lactamases differ from one
another in substrate profiles which depend on the number and types of
antibacterial agents they can inactivate. They also differ in terms of their
inhibitor profile. Moreover, the amino acid composition of these enzymes is
another factor in distinguishing the similarities and the existence of active
hydrolytic parts of the enzyme (Ambler, 1980; Bush, 2010 b). In Gram-
negative bacteria, the occurrence o f p-lactamase mediated resistance is either
expressed chromosomally or is plasmid borne. However, the spread of P-
lactamases is frequently associated with plasmid encoded ESBLs, specifically
the CTX-M family, and serine carbapenemases KPC and the Metallo-p-
lactamases (MBLs) VIM, IMP and NDM-1 (Pitout, 2010). Based on substrate
specifications, four major groups o f P-lactamases have been identified so far;
penicillinases, AmpC-type cephalosporinases, ESBLs and carbapenemases.
For the purpose of my thesis, I will primarily focus on ESBLs and
carbapenemases rather than the less-extended p-lactamases.
24
1.5.3.2 Classification of p-lactamases
The importance of the antibiotics penicillins and cephalosporins to treat
infectious diseases has led to the focus on exploring the characteristics of
enzymes produced by bacteria that hydrolyze these antibiotics. Many bacteria
are able to exhibit a new approach to withstand antibiotics, more specifically
(3-lactams. This is frequently noticed by the insertion of new nucleotide
sequences in the genetic context o f a particular antibiotic resistance gene or by
changing of one or more nucleotides in the nucleotide sequence that lead to
different amino acid sequences e.g. TEM group of P-lactamases.
Consequently, this may result in a different substrate hydrolysis profile that
can lead to a higher level of antibiotic resistance. However, a decrease in
antibiotic hydrolysis may also be observed. By 2009 more than 500 unique
protein sequences for p -lactamases had been reported (Bush & Jacoby, 2010).
P-lactamases have been classified in two ways, the first classification is
Ambler classification based on the classification of P-lactamases according to
their primary structure (Ambler, 1980), while Bush, Jacoby, Medeiros
classification is based on functional characteristics of p-lactamases (Bush et
a l , 1995).
1.5.3.3 Extended spectrum p-lactamases (ESBLs)
ESBLs are a group of enzymes able to hydrolyze and confer resistance to
penicillins cephalosporins, monobactams and oxyimino-cephalosporins that
include cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefepime. These
25
enzymes do not affect some cephamycins such as cefoxitin and cefotetan.
ESBLs have no or little activity towards carbapenems. They are inhibited by
the classical p-lactamase inhibitors; clavulanic acid, sulbactam and
tazobactam. The majority of ESBLs have been classified under Ambler class
A P-lactamases, these enzymes include blasnv and blajû that have evolved
from e.g. blasuv-i and bla^M-i encoding genes. Such derivation is attributed to
one or more point mutations occurring on the p-lactamase active site (Paterson
& Bonomo, 2005).
ESBLs are often found carried on large plasmids. In addition, a number of
antibiotic resistance genes that confer resistance to antibiotics such as
aminoglycosides and trimethoprim/sulphamethoxazole are also found on the
same plasmids. ESBLs are considered among the largest group of p-lactamase
known to activate antibiotics such as penicillins and cephalosporins rendering
carbapenems as the last choice for treating infections, this results in more
pressure on carbapenems. (Bush, 2010b). CTX-M enzymes are among the
wide spread ESBLs, since their first description in 1989 (Bauemfeind et al.,
1990), over 120 CTX-M type ESBLs have been discovered to date
(http://www.lahey.0 rg/studies/0 ther.asp#tablel). CTX-M ESBLs are grouped
into five major clusters; CTX-M-1,2,8,9 and 25 (Barlow et al., 2008 &
Bonnet, 2004), CTX-M 1 and CTX-M 9 being the most diverse clusters with
31 and 22 variants identified respectively. CTX-M enzymes comprise a wide
range o f subgroups for instance; CTX-M group 1 1, 2 and 9 are known to
26
include more members of CTX-M variants than CTX-M 8 and 25 e.g, CTX-
M -l,3,10,11,12,32,36 and CTX-M-15, CTX-M group 2 encompasses CTX-M-
2,20,31,5,6,56,7 and others, CTX-M-9 includes for instance; CTX-M-
9,13,14,17,47,48 and CTX-M-55. CTX-M groups 8 and 25 includes only few
variants (Novias et al., 2010 & Harada et al., 2008). The dissemination of
CTX-M ESBLs is oftentimes associated with the occurrence of Insertion
Sequence Common Regions (ISC7?s) which are found to be located upstream
of antibiotic resistance genes and can activate their transmission. The
occurrence of CTX-M ESBLs in E. coli isolates from nine patients in Norway
has been recently assessed. Six o f the ESBL genes were blacjx-M-is and one
Z?/tfcTx-M-3 - All blacix-M-\5 bore resemblance to each other in terms of their
sensitivity to antimicrobials used with minimum inhibitory concentrations
(MICs), > 256 pg/ml and >256 pg/ml for cefotaxime and ceftazidime,
respectively (Naseer et al., 2007).
1.5.3.4 Carbapenemases
Carbapenems are hydrolysed by carbapenemases produced by Gram-negative
bacteria such as members of Enterobacteriaceae and non-fermenters. These
enzymes have been classified into three classes according to Ambler
classification; class A, B and D. Class A and D are known as serine
carbapenemases and Class B are called metallo- p-lactamases (MBLs) (Walsh,
2010).
27
1.5.3.5 Class A carbapenemases
Class A carbapenemases are also known as group 2f, according to Bush et al.,
1995, comprises five phylogenetic groups; NMC, IMI, SME, KPC and GES
and are subdivided into chromosomally and plasmid mediated groups. SME,
NMC and IMI are chromosomally mediated whereas KPC and GES groups
are, in most cases, plasmid mediated. These enzymes possess hydrolytic
activity towards_most p-lactams including carbapenems, cephalosporins,
penicillins, and aztreonam and have been found in Enterobacteriaceae and P.
aeruginosa. SME-1, SME-2 and SME-3 were chromosomally mediated in
Serratia marcescens whereas IMI-1, IMI-2 and NMC-A are detected on the
chromosome of Enterobacter cloacae. GES-2 was found plasmid mediated in
P. aeruginosa, GES-4 has been detected on a plasmid in K. pneumoniae
isolated from a Japanese patient whereas GES-5 and GES - 6 were plasmid
mediated in E. coli and K. pneumoniae isolated from Greece (Queenan and
Bush, 2007; Walsh, 2010).
KPC enzymes are among the plasmid encoded class A serine carbapenemases,
mostly from K. pneumoniae, and are considered the most frequently detected
class A enzymes that have a potent threat to antimicrobials used to treat
infections. KPC enzymes were first discovered in K. pneumoniae isolated
from a patient from North Carolina, USA in 1996. KPC-1 was followed by
KPC-2 and, later on KPC3, KPC-4, KPC-5, KPC - 6 and KPC-7 as variants of
KPC-1 and KPC-2. KPC genes have been reported on plasmid in
28
Enterobacterial species; E. coli, Salmonella cubana, E. cloacae, Proteus
mirabilis, and K. oxytoca. Self transferable KPC genes have been determined
to be transferred to E. coli, they have also been detected carried on a lOkb
transposon, Tn4401, and associated with the insertion sequences \SKpn6 and
ISKpn7 (Nass et al., 2008; Nordmann et al, 2009; Walsh, 2010; Queenan and
Bush, 2007).
1.5.3.6 Class D p-lactamases
This class of p-lactamases includes enzymes called oxacillinases, these
enzymes hydrolyze cloxacillin, oxacillin, extended spectrum cephalosporins
and carbapenems. Oxacillinases such as blaoxA-\ and blaoxA-\o are among
enzymes that show increased hydrolysis of cloxacillin or oxacillin whereas
blaoxA-u and 6 /aoxa-i5 hydrolyse cloxacillin or oxacillin and even oxyimino-
p-lactams less efficiently than others. Some p-lactamases can target
carbapenems for instance blaoxA-23 and blaoxAAS in addition to cloxacillin and
oxacillin, these enzymes have been detected plasmid mediated in
Enterobacteriaceae (Poirel et al., 2004; Walther-Rasmussen and Hoiby, 2006).
Four clusters o f oxacillinases are responsible for carbapenem hydrolysis in
Gram-negative bacteria; blaoxA-23, blaOXA-2A, bla0xA-5s and bla0xA-4s (Walther-
Rasmussen & Hoiby, 2006). blaoxA-23 cluster comprises two enzymes; blaoxA-
27 and blaoxA-49. The majority of these enzymes are found in Acinetobacter and
can be chromosomally or plasmid mediated (Poirel & Nordmann, 2006).
29
1.5.3.7 Metallo-P-lactamases (MBLs)
MBLs are enzymes capable of readily hydrolysing all p-lactam antibiotics
with the sole exception of monobactams. In addition they are not inhibited by
the classical serine p-lactamase inhibitors (Walsh et al., 2005), (Jones et al.,
2005b; Poirel et al., 2010a; Samuelsen et al., 2010; Walsh et al., 2005). At
molecular level, MBLs are a disparate group of proteins, they are classified to
three classes; B l, B2 and B3 based on sequence identity and other structural
features. Classes Bl and B3 possess two zinc ions in their active sites and
class B2 possesses only one zinc ion. The widely spread enzymes belong to
class B l, these enzymes posses the key zinc coordinating residues of three
Histidine and one cysteine such as; IMP, VIM, GIM and SPM-1, class B2
include enzymes that posse asparagine instead of Histidine (Walsh et al.,
2005) Most MBL genes are located on mobile genetic elements, the majority
of these MBL encoding genes are carried in the form of gene cassettes on class
1 integrons and/or Tn402-type transposons (Marchiaro et al., 2010; Poirel et
al., 2010b; Borgianni et al., 2011; Lee et al., 2005; Castanheira et al., 2004;
Santos et al., 2010) whereas some o f these genes are associated with insertion
sequences such as ISCR4 (Z>/<2s p m - i ) , (Salabi et al., 2010; Poirel et al., 2004)
and IS2d/Tn5 transposon (Yong et al., 2009) which can facilitate their global
spread. MBLs have been reported worldwide in non-fermenting Gram-
negative bacteria (Osano et al., 1994) and more recently in Enterobacteriaceae
(figure 1.12) (Kumarasamy et al., 2010)
30
The continuous emergence of MBLs and their association with MDR
phenotypes in Gram-negative bacteria are considered major threats in the
treatment of infectious diseases. To date 9 acquired MBLs have emerged
worldwide (Figure 1.12); IMP (Osano et al., 1994), VIM (Lauretti et al.,
1999), SPM-1 (Toleman et a l, 2002, GIM-1 (Castanheira et al., 2004), SIM-1
(Lee et al., 2005), AIM-1 (Gupta, 2008), KHM-1 (Sekiguchi et al., 2008),
NDM-1 (Yong et al., 2009) and DIM-1 (Poirel et a l, 2010) genes in addition
to the novel TMB-1 that was recently detected in Libya (see chapter 6 ).
The prevalence of carbapenem resistance strains of P. aeruginosa has been
reported in China during the period of 2004 to 2005. P. aeruginosa strains
have been collected from different cities in China including a large teaching
hospital in Beijing and data shows that 10 % of all imipenem resistant P.
aeruginosa carry blayim type MBLs. 12 out of 14 strains of P. aeruginosa
were positive for class 1 integrons carrying blay\u-i- These results reveal that
blayim-2 type MBL genes disseminated horizontally in China between different
cities, due to patients transfer among cities inside the country. (Yu et al.,
2006).
Numerous strains of Gram-negative bacteria possess chromosomes that have
become a mosaic as a result o f the horizontal gene transfer and the vertical
inheritance of genes (Waldor, 2010). Four mechanisms by which antibiotic
resistance genes can horizontally be mobilised from a chromosome to a
31
plasmid are integrons, transposons, Integration Conjugative Elements (ICE)
(Partridge, 2011) and Insertion Elements (Toleman & Walsh, 2011).
A - SPM l
- GIM-l
A - AIM-1
- SIM-i
* - NDM-l
- KHM-1
Figure 1.12 Global emergence of MBLs (modified from Walsh, 2010) .
1.6 Global emergence of clinical antibiotic resistant Gram-negative

bacteria
Gram-negative bacteria, more importantly those belong to Enterobacteriaceae
and non-fermenters such as P. aeruginosa and Acinetobacter baumannii, are
among the most causative agents of hospital and community acquired
infections. Extended-spectrum cephalosporins, fluoroquinolones and
carbapenems are among the main therapeutic choices. The continuous pressure
of (3-lactam antibiotics in hospitals has exacerbated the selection for
consecutive generations of P-lactamases - ESBLs followed by
carbapenemases (Chouchani et al., 2011; Coque et al., 2008). The problem
32
becomes worse when such resistance occurs by horizontal gene transfer or
mediated by conjugative plasmids as it is the case generally for ESBLs
causing resistance to extended-spectrum cephalosporins. For example, CTX-
M-type ESBLs have been detected in Europe with ^/tfcrx-M-is, ^^ctx-m -3 and
blacYx-M-9 carried on a variety of different Inc-type plasmids and sizes (Figure
1.13) (Livermore et al., 2007).
D om inant enzym e O th e r freq u en t types
CT X -M -15 • C T X -M -15
® C T X -M -3
C T X -M -3
C T X -M -1
C T X -M -9
O C T X -M -10
A C T X -M -9
A C T X -M -14
Figure 1.13 The emergence of CTX-M type ESBLs in Europe.

(Livermore et al., 2007)
According to the antimicrobial resistance surveillance conducted in Europe
between 2006-2009, the recent global emergence of antimicrobial resistance of
K. pneumoniae, E. coli and P. aeruginosa in Europe showed that there is an

increasing trend in the resistance of these micro-organisms to antimicrobials
used. The surveillance showed that E. coli isolates collected from European
countries exhibited high resistance to aminopencillin, extended-spectrum
cephalosporins and aminoglycosides. European antimicrobial resistance
surveillance (EARS-Net) data also shows a continuing increase in
flouroquinolone resistance. High proportion (85-100%) of E. coli isolates
resistant to extended-spectrum cephalosporins were due to ESBLs indicating
the high prevalence of ESBL producing E. coli in European hospitals
(http://ecdc.europa.eu/en/publications/Publications/101 l_SUR_annual_EARS
_Net_2009.pdf).
A high proportion of resistance of K. pneumoniae to extended-spectrum
cephalosporins, fluoroquinolones and aminoglycosides is evident. K.
pneumoniae isolates from two countries; Greece and Cyprus in the
Mediterranean Gulf also show high resistance to carbapenems. Half of the
countries involved in the surveillance program reported the incidence of multi
drug resistant (MDR) K. pneumoniae to extended-spectrum cephalosporins
(Figure 1.14), aminoglycosides and fluoroquinolones whereas northern
European countries such as Denmark and Norway reported an increasing trend
of resistance to specific classes of antibiotics whilst emergence of resistance in
UK showed a consistent reduction (http://ecdc.europa.eu/en/publications
/Publications /1011_SUR_ annual_ EARS_Net_2009.pdf).
34
With respect to EARS-Net data on P. aeruginosa in Europe, data on the
resistance trends from the eastern and southern parts of Europe show a higher
proportion of antibiotic resistance. Overall, of 8129 P. aeruginosa isolates
collected from the 28 countries participating in the surveillance, 1541 have
shown resistance to carbapenems; imipenem and meropenem (Figure 1.15).
(http://ecdc.europa.eu/en/publications /Publications /1011_SUR_ annual_
EARS_Net_2009.pdf).
Carbapenems were introduced as a first line therapy to treat infections caused
by non-fermenters in the 1980s, they have also been used for ESBL-producing
Enterobacteriaceae after the increasing trend of resistant enterobacterial
species to 3rd generation cephalosporins. Since then acquired carbapenemases
started to appear and attracted increasing attention most notably MBLs and to
lesser extent other carbapenemases such as class A. Since the discovery of
MBLs, 9 of these enzymes with their variants have been reported in Latin
America, USA, Europe, Africa, Southern Asia, India and Australia and
recently TMB-1 in Libya (see chapter 6 ). Recently MBLs were found in K.
pneumoniae, E. coli and E. cloacae such as Zj/andm-i that first emerged in
India, followed by the UK, and currently has been detected in many countries
worldwide (Pfeifer et al., 2011; Chen et al., 2011; Wu et al., 2010; Perry et
al., 2011; Sole et al., 2011; Jovcic et al., 2011; Yamamoto et al., 2011).
35
■i<t%
CIS 1% to <5%
d D 5% lo < 10%
d H O % to < 2 5 %
■ ■ 25% to <50%
n*50%
r l No data reported or less than 10 isolates
C d Not included
Non-visible countries
■ ■ Luxembourg
mm Malta
Figure 1.14 The occurrence of K. pneumoniae resistant to 3rd generation

cephalosporins in Europe (http://ecdc.europa.eu/en/publications/Publications
/1011_SUR_ annual_ EARS_Net_2009.pdf)
36
■ ■ < 1%
d a 1% to <5 %
e r a 5% to <10%
r ~ i 10% to < 25%
■ ■ 25% to < 50%
■12)0%
d a No data reported or less than to Isolates
I I Not included
Non-vislbte countries
BM Luxembourg
I S Malta
Figure 1.15 The occurrence of P. aeruginosa resistant to carbapenems in

Europe(http://ecdc.europa.eu/en/publications /Publications /1011_SUR_
annual_ EARS_Net_2009.pdf)
1.6.1 Evolution of antibiotic resistance in Gram-negative bacteria
Antimicrobial resistance surveillance programs are vital and considered a
longitudinal means of detecting changes in resistance to antimicrobials in
clinically important pathogenic bacteria. These programs include the
Meropenem Yearly Susceptibility Test Information Collection (MYSTIC)
(Turner et al., 1999), SENTRY (http://www.jmilabs.com/surveillance/),
Intensive Care Antimicrobial Resistance Epidemiology (ICARE) (Perasso et
al., 1999), European Antimicrobial Resistance Surveillance (EARSS)
37
(http://www.hps.scot.nhs.uk/haiic/amr/earsurveillance.aspx) and others. The
monitoring of these programs provides information on the increasing or
decreasing level of antibiotic resistance rate worldwide. It moreover offers a
guide for empirical treatment regimens. The massive use of antimicrobial
agents is the leading cause o f the prevalence of antibiotic resistant strains in
community and hospital settings.
As an example, a longitudinal study was carried out from 1993 to 2004 aimed
to assess the resistance rates o f Gram-negative bacilli that cause infections in
the intensive care units in the United States (Lockhart et al., 2007). Forty three
US states in addition to Columbia were included in this study and 74,394
isolates belong to 11 species of Gram-negative bacteria were collected and
tested against 17 antibiotics. The results showed that 22.2 % of all Gram-
negative isolates were P. aeruginosa followed by 18.8 % E. coli and 14.2 %
K. pneumonia, with additional low percentages of other Gram-negative
bacteria. Furthermore, P. aeruginosa was the highest among UTIs with 29.9
%. E. coli represented the highest among urine isolates with 42.4 %, while it
counted as 23.9 % in the blood. Antibiotic susceptibility testing revealed that
the highest resistance rate have been recorded for ampicillin-sulbactam, with
five-fold increase in the resistance of P. aeruginosa, while evaluation of the
rate of multi-drug resistance between 1993 and 2004 showed that a
longitudinal increase in MDR has been observed (Table 1.3). It has been
noticed that there is an association between fluoroquinolone usages as a
38
therapy and resistance, because the prolonged use of these antibiotics have
attributed to the rise of ESBL producing E. coli and P. aeruginosa. (Lockhart
et al., 2007). According to the CDC, MDR is defined as the resistance of
bacteria to > 3 classes o f antibiotics.
Table 1.3 Longitudinal increase in multi-drug resistance in USA

(Lockhart et a l 2007)
1993 2004
Organism
No. o f MDR No. of MDR % of
% o f MDR
isolates/total no. isolates/total no. MDR
isolates
of isolates of isolates isolates
P. aeruginosa 13/769 1.7 93/1004 9.3
E. coli 0/724 0 16/808 2
K. pneumoniae 26/513 5.1 84/633 13.3
E. cloacae 13/397 3.3 24/406 5.9
Acinetobacter spp. 19/285 6.7 101/338 29.9
E. aerogenes 6/213 2.8 0/154 0
P. mirabilis 1/174 0.6 1/142 0.7
C. freundii 5/95 5.3 7/63 11.1
39
Another study conducted in Sierallana Hospital in Spain sought factors that
may have an additional effect on patients admitted with bacteraemia. Blood
samples from 15045 patients were collected to determine the causative agents
of bacteraemia in the period from 1997 to 2005. Antibiotic susceptibility tests
were performed using the following antimicrobials; ampicilin,
amoxicillin/clavulanate, pipracillin/tazobactam, cefotaxime and trimethoprim/
sulfamethoxazole. 14.9 % of the patients had positive blood cultures, of
which, 4.4 % of isolates were E .coli. It has been reported that the factors that
attributed to the occurrence of bacteraemia in this hospital were; MDR E.coli,
ESBL producing E. coli, age of patients, time of treating with antibiotics and
the presence of severe sepsis, which collectively had a role in the morbidity
due to E. coli infections (Peralta et al., 2007).
The activity of meropenem and 11 other antimicrobial agents including third
generation cephalosporins has been assessed in the USA for 10 years in the
period between 1999-2008 to demonstrate any increase or decrease in the rate
of antibiotic resistance. A steady increase in the resistance rate of
ciprofloxacin was observed among E. coli (Figure 1.16), an increase in the
resistance of K. pneumoniae strains was detected for meropenem, ceftazidime,
piperacillin/tazobactam, tobramycin and ciprofloxacillin from 2004 to 2007,
however the resistance rate to these drugs slightly decreased in 2008 (Figure
1.17). (Rhomberg and Jones, 2009)
40
35
30
CC 20
15
VJ „_
® 10
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
Study year
■-a Meropenem » Ceftazidime ♦ Piperacillin/Tazobactam Tobramycin » - Ciprofloxacin
Figure. 1.16 Annual rate of antimicrobial resistance among E. coli

isolates (4394 strains) tested against selected agents from the MYSTIC
Program (1999-2008). (According to (Rhomberg and Jones, 2009)
25
ou>
c
I35
K
1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
Study year
A Meropenem —♦-Ceftazidim e ♦ Piperacillin/Tazobactam —JK—Tobramycin — Ciprofloxacin
Figure. 1.17 Annual rate of antimicrobial resistance among A.

pneumoniae isolates (2694 strains) tested against selected agents from the
MYSTIC Program (1999-2008). (According to Rhomberg and Jones,
2009)
41
In Arabia, data on antimicrobial resistance is lacking. However, in Tunisia the
appearance of blacix-u family occurred in 2005 after the identification of
blacix-u-2i associated with ISEcpl in Salmonella enterica and continued to
appear in 2006, 2009 and 2010. MBLs were only found in three isolates,
blayim-2 in two isolates of P. aeruginosa and blaym-A produced by K.
pneumoniae in addition to other ESBL genes. Oxacillinases started to be
reported in 2007 when blaoxA-\% was detected in P. aeruginosa and different
OXA enzymes continued to emerge up to the discovery of blaoxA-4 & the
carbapenem hydrolysing enzyme in K. pneumoniae in 2010. (Figure 1.18)
(Chouchani et al., 2011). The first report of blaax-MA5 and blacix-uA in E.
coli, K. pneumoniae and E. cloacae isolated from two hospitals in Bejaja,
Algeria appeared in 2006 (Touati et al., 2006) followed by detection of blacxx-
m -15 in K. pneumoniae and E. coli from hospital environment (Touati et al.,
2007) and in Salmonella enterica isolated from patients in Algeria (Touati et
al., 2008). ^/aviM-i9 was reported as a novel MBL found in Enterobacteriaceae
in Algeria (Robin et al., 2010). Mechanism of antibiotic resistance in clinical
isolates of P. aeruginosa from patients admitted to the University affiliated
hospital of Tlemcen in Algeria was due to the production of blaoxAAO and
blajûwo (Drissi et al., 2008). The first description of CTX-M producing
Gram-negative bacteria in Egypt was from clinical isolates of E. coli in 2006
(Mohamed Al-Agamy et al., 2006) while the first report of 6/<3tem and 6/ashv
appeared in 2009 (Ahmed et al., 2009), showing the lack of research on this
subject. ^/«ndm-2 was the only MBL detected in A. baumannii from Egypt
42
(Kaase et al., 2011). Furthermore, work on bla0x a enzymes from Egypt
appeared only in 2011 (Ahmed and Shimamoto, 2011). Plasmid mediated
Z>/#tem-3 has been detected in S. typhimurium isolated from patients admitted to
the IbnRochd University hospital of Casablanca (AitMhand et al., 2002),
moreover, blarEM and blasnw were reported from E. coli and K. pneumoniae
isolated from community acquired urinary tract infections from three
Moroccan cities; Casablanca, El Jadida and Settat (Barguigua et al., 2011).
Research on ESBLs, oxacillinases and MBLs started to appear in 2011
showing the lack of focus on antibiotic resistance in Gram-negative bacteria
(Barguigua et al., 2011; Porton et al., 2011 & Poirel et al., 2011).
1994:TEM -1
1998: CMV-2 ,CMV-4
1999: TtM-1; SHV-2; Amp-C {noil identified)
M editerran ean S ea 2003: TIM-4;SHV-2a;ACC-la
TEM-4;SHV-2a;ACC-la
2004: SHV-12,-SHV-2a
SHV-12;SHV-2a
2006: TEM-138; CTX-M-15.-CTXM-16
CTX-M-15;CTX-M-16
2007: TIM-15 ;OXA-18
2008: OXA-1;Tt M-1;SHV-1;SHV-11 ;SHV-27,'SHV-103;CTX-M-l 5
2006:OXA-l;TTM-l;SHV-l^HV-U;SHV-27,-SHV-103;CTX-M-lS
- 2009: TIM-164; CTX-M28;CTX-M-9; OXA-18;SHV-2a;SHV-S;5HV-12
2010: CTX-M-l.-THM-lb.Tf M-20.-CMV-2; VTM-2;SHV-2a
2005: CTX-M-27
2008: OXA-69; OXA-23; OXA-97
2009: CTX-M-15;SHV-2a;SHV-12£HV-28;TEM-l;LAP-2; VIM-2
2010: CTX-M-153HV-12,-SHV-2a
2010: CTX-M-15,-CTX-M-14,-(TX-2 7;SHV-12;SHV-2a
1991: SHV-2
2002: ACC-1
2006: ACC-1; VIM-4 ;CTX-15 ;CMY-4
2009: TEM-l;SHV-2a ACC-1
2010: CTX-M-lS,-SHV-12,-SHV-2a
Figure 1.18 The occurence of p-lactamases, ESBLs and carbapenemases

in Tunisia (Chouchani et al., 2011)
43
1.7 DNA structures that spread antibiotic resistance
1.7.1 Plasmids in multi-resistant Gram-negative bacteria
Plasmids are extra-chromosomal DNA found in the cytoplasm of bacteria as
independent genetic moieties capable of autonomously reproducing copies of
the same plasmid within the cell in the presence of mechanisms to control
plasmid copy number and the stability of plasmid inheritance. Plasmids carry
essential genes for establishing and directing replication. Furthermore, they do
not normally have any functional contribution that is necessary for the cell or
cell growth. Plasmids are circular and sometimes linear double stranded DNA
segments that normally replicate without affecting the circular chromosome
(Carattoli et al., 2005). Plasmids are known to carry genes code for
detoxification, ecological interactions, virulence and antibiotic resistance.
Plasmids can confer and mobilize resistance to antimicrobials by acquiring
resistance genes via horizontal gene transfer and consequently increase the
genetic diversity of bacteria.
Resistance genes in Enterobacteriaceae have different constraints for host
ranges depending on the plasmids that carry them. It is supposed that genes
carried on IncP, IncA/C and IncQ can move to genera of Enterobacteriaceae in
addition to Pseudomonas and even Gram-positive bacteria due to their larger
host range. Other plasmids such as IncFII have a limited host range restraining
the transferability of antibiotic resistance genes located on these plasmids, for
instance blacix-u -\5 does not have the ability to move to non-fermenters such
44
as Acinetobacter and Pseudomonas and only limited for Enterobacteriaceae
(Carattoli, 2009; Smillie et al., 2010).
1.7.2 Pathogenicity islands (Multi resistance in bacteria)
The multi-resistance genotype can reflect the occurrence of resistance islands
that include a considerable number of resistance markers and are known as
genomic islands (Schmidt & Hensel, 2004). Several bacterial species have
shown that the multi-drug resistance phenotypes were mainly attributed to the
incidence of resistance islands. These isolates include; Shigella flexneri, S.
enerica, Vibrio cholera and Staphylococcus aureus with genomic islands sized
20 to 60 kb (Dobrindt et al., 2004).
One of the first resistance islands to be fully characterised from genomic
sequencing was that by Fournier et al. that reported an 86kb island from A.
baumannii. This strain, AYE, included 45 antibiotic resistance genes, 25 of
which belong to p-lactams, aminoglycosides, fluoroquinolones, tetracyclines,
trimethoprim, chloramphenicol, rifampicin and sulphonamides. Several
antibiotic resistance genes were previously reported in Acinetobacter spp for
instance 6 /tfo x A -io and blaye b - i , aac3, aadA\/B and dhfrl, were also found in
this island whereas some other resistance genes had not been reported in
Acinetobacter species before such as aac6, tetA, cmlA, dfrX and blaoxA-69.
aac6 confers resistance to aminoglycosides except gentamicin, tetA is a
tetracycline resistance gene, cmlA encodes the multidrug efflux pump
Cmr/MdfA, dfrX confers resistance to trimethoprim and blaOXA-69 is a class D
45
(3-lactamase found to weakly hydrolyse imipenem and meropenem (Figure
1.19). The island also showed the incidence of three class 1 integrons with 14
gene cassettes embedded within these integrons (Fournier et a l 2006).
Transposons and insertion sequences were detected in the island and showed
the occurrence of 22 ORFs encoding transposases, 4 transposons, 2 truncated
transposons Tn5393 and Tnl72J and two Tn-like transposable elements. The
occurrence of this massive number of antibiotic resistance markers, antiseptics
and mercury resistance genes in one strain shows how complex the genetic
pool can be. (Fournier et al., 2006).
f
ooaa Irxntpovuor. vAA
W po fwttbvc
^rvB
tffraroJovrn
m
.»r»H
<m m
anC
m
« rC | /> * « Hcav> mco> ftp * In a n w o o
L----- ^ k tA u fM K w w n p ra -------------- -— — . 2
urA kfffl
■ p m K> <33 L 3 <53 Cd (1

a* MR m A / vaR Iiiw p q w n < O i r f l - i M w a w
MBKW9 rcMSBMMV oparon
G5E> Lit
a e > i~ > < * g >
TnpA mrCy * '« f •rn.tr wmA m R j *•/.* W .M T.vA i; ‘ NA wW, M fla l)
1«C/ h i* t w u j i m a t r e a t e d Tn! '; t like mn$po*c* IS / Nfc* tssmswreon
G
><3 <3 G
>D <3 E>OE>€3aE»DDC3C3>»
I S /-1 T rto v M M f R tt« lv u c IS /5 I S 4 / M i IS.'* inpM ! Ml « rf\ orfX urDT o u iH I
.WW»N'/Itf *>p
*WI orlS : nM J ,,KAt H . , Ym . I /.r A [•

Best Blast mcndi with
(c fit
wflmwnnnrc
Figure 1.19 Genomic island of A. baumannii AYE according to (Fournier,

PE. et a l , 2006)
46
1.7.3 Transposons
Some transposons contribute to the movement of antibiotic resistance genes as
part of class 1 integrons. These transposons include the Tn3 family, the
Tn5053 family and Tn402-like transposons. These families differ from each
other in terms of structure and transposase genes carried by these transposons.
The Tn3 family is composed of two subgroups; Tn3-like and Tn27-like
transposons, they share the same 38bp Inverted Repeat IR, transposase gene
(itnpA), a resolution site (res) and resolvase gene (tnpR). These transposons
carry antibiotic resistance genes as part of class 1 integron, moreover, they
carry mercury resistance genes and genes specific for transposition functions.
Unlike the T ni family of transposons, the Tn5053 family and Tn¥02-like
transposons are responsible for carrying and spreading antibiotic resistance
genes captured by class 1 integrons. Two major steps have been proposed to
elucidate the mechanism by which class 1 integron has become part of the
Tn-702-like transposons. The first step was by inserting the integron inside the
Tn402-like transposons while the other step suggest the formation of the
conserved segment (qacEA/suII) followed by the loss of part of tni. (Toleman
et al., 2007; Sajjad et al., 2011). Tm/02-like differ from the Tn3 family in
having three transposase genes; tniA, tniB, tniQ, the resolution site res is
located between these genes and the resolvase gene tniR and sometimes called
tniC gene. Tn402-like transposons are increasingly reported carrying antibiotic
resistance genes in the form of gene cassettes embedded in class 1 integrons.
(Partridge, 2011).
47
Transposons such as Tn5090/Tn402 carrying blavm -2 was detected in a
clinical isolate of Indian P. aeruginosa. Sequencing of the full transposon,
including the integron showed that this structure is very much like the
American and Russian blay im -2 integron structures harbouring aacA 7, 6 /< 2 V im -2 ,
dhfrB5 and tniC. All three integrons had the same variable region structure
and lacked the conserved segment considered a character of class 1 integrons
harboured by TN5090/Tn402 transposons, resulting from excision and
acquiring of gene cassettes. (Toleman et al., 2007).
1.7.4 Integrons:
Integrons are genetic elements found in most cases, plasmid mediated and
recently some large integrons were detected on the chromosome. Integrons
carried on plasmids are responsible for the incorporation of antibiotic
resistance genes known as gene cassettes inside the integrons and as a result of
this integration they enhance the expression of the gene conferring resistance
to antimicrobials. (Walsh, 2006; Mazel, 2006). The first integron was detected
in Gram-negative bacteria as a mechanism by which integrons in cooperation
with transposons can express multi-resistance phenotype. Integrons are
classified in two kinds; mobile integrons and superintegrons. Mobile integrons
are always plasmid located and are divided into five classes; class 1 integrons
originated from Tn402 and is found inserted in Tn27 (mazel, 2006). Class 1
integrons are largely associated with acquiring and mobilising antibiotic
resistance genes and are counted as the main responsible system for such
48
occasion in Enterobacteriaceae. The wild-type class 1 Integron is composed of
two sequences; 5 conserved sequence which is also known as (5'CS) and 3
conserved sequence (3'CS) where 5'CS represents the Intgrase gene and 3'CS
comprises quaternary ammonium compound resistance gene (qacAEl) and
sulphonamide resistance gene (sul 1), respectively. (Cambray et al., 2010).
This class of mobile integrons is responsible for conferring resistance to some
p-lactams such as aminoglycosides, trimethoprim, rifamycin, erythromycin,
streptothricin, chloramphenicol, fosofomycin, quinolones and antiseptics. The
integrase (Inti) gene is the functional constituent of the integron; it encodes an
enzyme called site-specific tyrosine recombinase and it operates to excise and
integrate gene cassettes on the attachment site (<attC). It is called
recombination process (Walsh, 2006). The majority of gene cassettes are
promoter-less and requires Pc promoter embedded on the integrase gene or attl
site. (Cambray et al., 2010).
Class 2 Integrons are found embedded on large transposon called Tn7 and
despite the fact that class 2 integrons encode for non-functional proteins due to
a nonsense mutation in codon 179, they are likely to confer resistance to six
antibiotics, whereas Class 3 integrons are less frequent than class 2 integrons.
Class 4 and 5 are mainly related to trimethoprim resistance in V. cholerae.
Super Integrons are larger than mobile Integrons and they have been described
in V. cholerae and because of their location on the chromosome; alone, they
49
are not mobile and consequently are not capable of mobilising genes (Mazel,
2006).
Class 1 integrons in particular play an important role in disseminating
carbapenemase encoding genes such as MBLs in addition to other antibiotic
resistance determinants in Enterobacteriaceae and non-fermenters. The most
virulent and crucial factors for high levels of resistance to carbapenems
particularly MBLs were identified as carried on class 1 integrons, for instance;
bla^dm-i? blctyim-1? blci\jiM-2 >bloiMP and bladim-i (Poirel et al., 2010; Yong et
al., 2009; Walsh et al., 2005; Zhao et al., 2009). Class 1 integron was also
found to have contributed to dissemination of antibiotic resistance genes to
unrelated clinical isolates in Brazil where it has been detected carrying bla\u?-\
and a new aminoglycoside resistance gene, aac(6)-31 in P. putida, different
isolates of A. baumannii and Acinetobacter sp.(Mendes et al., 2007)
Integrons have also been detected in bacterial strains collected from manured
soil with increased prevalence o f integrons after slurry application. Class 1 and
2 integrons were determined in Acinetobacter, Aerococcus, Bacillus,
Enterococcus, Pseudomonas and Enterobacteriaceae (Byme-Bailey et al.,
2011). Class 1 integrons were also identified in sewage treatment plants
occurring at different levels in affluent water, activated sludge and effluent
water. It has been shown that 57 isolates out of 189 isolates belonging to E.
coli, Klebsiella, Aeromonas salmonicida, A. veronii and A. media, were
identified carrying class 1integrons (Ma et al., 2011).
50
1.7.5 Insertion Sequence Common Regions (ISCRs)
Common Regions (CRs) have been discovered since the mid-1990s as being
associated antibiotic resistance genes. It has a size of 2154 bp and
incorporated an open reading frame, orf513, that was found inserted adjacent
to class 1 integron and beside the sull gene (Toleman et al., 2006a). They
comprise orf575 and 33 bp sequence of DNA and they argued that it might
play a role in what is called recombination crossover site (RCS). Common
regions can promote the expression of some resistance genes in E. coli, K.
pneumoniae and A. baumannii and these genes are: qnrA, blacix-u-% blaax-u-
2 and dfrA10 (Rodriguez-Martinez et al., 2006).
Common Regions or Insertion Sequence Common Region (ISCR) have now
become an established mechanism of gene movement. It is proposed that ISCR
possess two ends, or/IS and terlS as insertion and termination sites of ISCR,
respectively. These insertion sequences have been found as truncated parts at
the right side of 3 'CS of class 1 Integron and associated with two genes; qac
and sul. Furthermore, this insertion sequence has been found without terlS -
providing evidence that a deletion event occurred. Misreading of terlS and
passing through many events of transcriptions and translocation resulted in the
development of these “complex class 1 Integron”, together with misreading
and homologous recombination has resulted in genes qac and sul added to the
end of 3 'CS (Toleman et al., 2006a).
51
Many derivatives of ISCRs have been discovered and associated with the
mobilisation of antibiotic resistance genes. These insertion elements were
firstly described in In6 and In7 class 1 Integrons. ISCR1 can carry
trimethoprim resistance genes such as dfrA23 and dfrA 18, also they have been
found to be associated with quinolones resistance (Stokes et al., 1989). ISCR1
has been detected upstream of qnrA in class 1 Integrons and virtually all
isolated genes were identical in spite of their country of origin. ISCR\ plays a
major role in the resistance of Gram-negative bacteria to aminoglycosides
where it has been detected upstream of arm A genes. Additionally, ISCRl is
associated with ESBLs that inhibit the activity of the antibiotic cefotaxime and
class A p-lactamases such as blay e b - 3 , 6 /« p e r-3 , blacuY-\ and blac m y - 9 - ISC/? 2
is also widely disseminated and associated with resistance islands such as SXT
via orfA. Despite the fact that ISCK2 is not associated with class 1 integrons, it
has been found associated with su ll gene. ISCR3 seems to be more specific
for Salmonella genomic island 1 genetic element (SGI 1 element) and
erythromycin gene (erm). However, it has also been proposed that it is
associated with the resistance of Stenotrophomonas maltophilia to
trimethoprim/sulfamethoxazole. (Toleman et al., 2006b)
1.7.6 Insertion Sequences
Insertion sequences are considered as the simplest bacterial mobile DNA
interms of their structure, they comprise more than 19 families, they have
different sizes but in general they range between 600 to 3000 bp. They consist
52
of one or more open reading frame that code for transposase proteins flanked
with short sequences of inverted repeates (Wagner et al., 2007) ISEcplB is
another paradigm of insertion sequences that is associated with mobilisation
and expression o f some antibiotic resistance genes. It is characterised by
several features; it can express and mobilise as well as disseminate the
cefotaxime resistance gene blacix-M-\9 - Promoter sequences, which are located
close to its inverted right repeat (IRR), can also facilitate expression of genes.
ISEcplB has been found associated with blacix-u -\9 in a strain of K.
pneumoniae resistant to ceftazidime (Poirel et al., 2003). Lartigue and
colleagues described the ability o f ISEcplB to mobilise and express the p-
lactamase gene, blacjx-M from a transposon, which was located on a
chromosome of Kluyvera ascorbata and moved to a plasmid (Lartigue et al.,
2006).
1.7.6.1 Integrative and Conjugative Elements (ICE)
Integrative and conjugative elements (ICE) are mobile genetic elements
known as self-transmissible and found in Gram-positive and negative bacteria.
ICE can be transferred from one strain to another by conjugation and lateral
gene transfer. Like plasmids and phages, ICEs compromise of three modules
divided according to functions responsible for maintenance, dissemination and
regulation. ICEs maintain their virtual inheritance by integrating into a
replicon of the host either plasmid or chromosome by means of gene encoding
a recombinase called Int that catalyze attP on the ICE and a target sequence
53
attB on the chromosome. ICE, on the circular form, can be integrated into the
chromosome by recombination between attP and attB, creating two ICE
chromosome junction sequences, aatL and attR. ICEs are excised by
excisionases called Xis and require the presence of attL and attR to perform
excision. Dissemination of single stranded DNA of ICE is carried out by
conjugation, genes specific for synthesis of mating machinery to enable the
initiation between donor and recipient cell to deliver DNA to the recipient cell.
(Wozniak & Waldor, 2010; Burrus & Waldor, 2004).
54
1.8 Objectives of study
Libya is located in North Africa bordered by the Mediterranean Sea from the
north, Egypt from the east, Sudan from the southern east, Chad and Niger
from the south and Algeria and Tunisia from the west. Libya is considered a
very rich country; it has one of the most important resources worldwide, oil.
Compared with other Arabic, European and Asian countries, Libya should
have been one of the best countries in terms of development, infrastructure,
investments and education; however, the last 40 years obfuscation and perfidy
have retarded this potential.
Given that there is little information known on antibiotic resistance in Gram-
negative bacteria in Libya and North Africa, I took the opportunity to
investigate the mechanisms of antibiotic resistance in Gram-negative bacterial
isolates collected from clinical, non-clinical and environmental settings in
Tripoli and Benghazi, Libya.
The study focuses on the characterisation of antibiotic resistance mechanisms
of isolates collected from patients admitted to different wards, in particular
Intensive Care Units (ICUs). Moreover this study investigates the spread of
MDR bacteria in the hospital environment to understand the occurrence of
outbreaks within an individual hospital or among different hospitals. The
emergence of MDR bacteria in non-clinical samples was also investigated in
this study in order to associate the spread of nosocomial pathogens among
55
patients, within the hospital and outside the clinical settings. This is the first
molecular study conducted on antibiotic resistance on bacterial strains isolated
from Libya and will hopefully provide a useful insight on the problem of
antibiotic resistance in Libya and in general Arabia.
56
Chapter Two
Methods and Materials

2.1 Bacterial collection.
Isolates used in this study were collected randomly from Tripoli and Benghazi
from hospitals and environment outside the hospital during 2008-2009.
Collection of the clinical samples included specimens from inpatients;
outpatients and the hospital environment. Environmental swabs were collected
from Tripoli and Benghazi streets, cafes etc. whereas the hospital environment
samples refer to swabs collected from hospital floors, comers, toilets, walls,
bedsides, sinks, curtains, trolleys, gauze containers, work tables and medical
devices such as; mechanical ventilators, oxygen cylinders, baby incubators,
nebulizers, anaesthesia, hypolizer, suction machine, tip of catheter. Bacterial
isolates cultured from the swabs were identified by the use of Phoenix (Becton
and Dickinson, USA). E. coli topolO kit (Stratagene, Amsterdam, the
Netherlands) was used in the cloning experiments. The swabs were transferred
to the lab in transferring charcoal media (Technical Service Consultants Ltd,
Heywood, UK).
2.1.1 Ethical considerations
The limited amount of information required for each specimen was such that
ethical approval was not considered necessary and because there is no ethical
board in Libyan hospitals.
58
2.2 Safety considerations
Regulations and safety were undertaken according to the Ionising Radiation
Regulations, 1999.
2.3 Bacterial strains used
The following bacterial strains were used in cloning experiments
Strain Genotype Reference/Source
F‘(|>80/tfcZAM 15A(/acZYA-
<zrgF)U 19 rec A 1 end A 1
D H 5a-Tl R Invitrogen Ltd
h s d R llfa , mk+)p h o A supE44
thi-\ gyr A \9 relA l ton A
Nordmann et al.,
E. coli J53
2008
A modified Escherichia coli
HB101 (UAB190) was used as
the recipient strain [rifampicin

E. coli GFP Mata etal ., 2011
and aminoglycoside resistant
and green fluorescent
protein (GFP) producing].

Pseudomonas
Barkay et al.,
aeruginosa
1993
PA01
2.4 Chemicals, reagents, and Radioactive labels.
Chemicals were purchased from BDH Chemicals Ltd and Sigma. Media
constituents were obtained from either Oxoid laboratories or Fisher Scientific
59
laboratories. Radiolabelled Phosphorus 32P was supplied from PerkinElmer,
Boston, MA02118, United State of America (USA), 800-762-4000, Random
primer labelling kits were supplied from Agilent Stratagene products, USA.
PCR Gel extraction kits and plasmid miniprep purification kits were supplied
from QIAGEN GmbH, D-40724 Hilden, Lambda Ladder PFGE Marker was
obtained from New England Biolabs.Inc. Digestive enzymes; Xbal, S 1 and
Spe 1 were purchased from Fermentas Life Sciences company. PCR Master
Mix was supplied from Thermo Fisher Scientific ABgene House, Blenheim
Road, Epsom, Surrey, UK.
2.5 Growth Media.
2.5.1 Luria Bertani Broth
L.B. broth was made up according to the manufacturer’s instructions (Fisher
Scientific Ltd).
2.5.2 Luria Bertani Agar
L.B. Agar was made following the manufacturer’s instructions (Fisher
Scientific Ltd).
2.5.3 Mueller-Hinton Agar
MHA was supplied by Oxoid Ltd plate poured ready to use in Etest
experiments
60
2.5.4 MacConkey Agar No.3
MA no.3 was used to distinguish phonotypically between K. pneumoniae and
E. coli in conjugation experiments, the medium was made up according to the
manufacturer’s instructions (Oxoid Ltd).
2.5.5 MacConkey Agar
MA was purchased from Oxoid Ltd plate poured ready to use.
2.5.6 MacConkey Agar for isolation of ESBL/MBL positive isolates
MA was made up and supplemented with 10mg/l of ceftazidime to be used as
selective media; preparation of media was carried out according to the
manufacturer’s instructions (Oxoid Ltd).
2.5.7 S.O.C Medium
This was used as part of the TOPOIO cloning kit purchased from Invitrogen,
Life Technologies, Carlsbad, California, USA.
2.6 Sterilisation of Media.
Media was sterilised by autoclaving at 0.75kg cm' for 20min at 121 °C.
2.7 Isolation of environmental strains
Swabs collected from non-clinical settings and the environment outside the
hospitals were cultured on MacConkey agar supplemented with 10mg/l of
ceftazidime to select for isolates resistant to third generation cephalosporins.
61
Pure cultures were obtained by sub-culturing mixed cultures from the primary
MA plates on a new selective MA plates supplemented with the same
concentration of antibiotic.
2.8 Etest experiments
Etest strips containing imipenem (IP) and EDTA as MBL inhibitor (IPI) were
purchased from. (BioMerieux, Paris, France). They were used to detect the
occurrence of metallo-p-lactamases (MBLs) in carbapenem resistant isolates.
2.9 Antimicrobial Susceptibility Testing and MIC determination
Antibiotic resistance profile tests and minimum inhibitory concentration
(MIC) determination for clinical, non-clinical and environmental isolates were
performed according to the Clinical Laboratory standards Institute (CLSI) by
the use of Phoenix 100 (Becton-Dickinson, Oxford, UK). MIC 50 and MIC90
were defined as the minimal concentration that inhibited 50% and 90% of
bacterial growth (Hsu et al., 2011).
2.10 Phenotypic and Genotypic Detection of ESBLs
2.10.1 Amplification of DNA sequences using the Polymerase Chain
Reaction (PCR)
2.10.1.1 Amplification of blacTx-M tyPe ESBLs
K. pneumoniae and E. coli isolates were screened for the occurrence of
blact x - m type ESBLs that belongs to the phylogenetic groups, 1, 2, 8, 9 and 26
62
using multiplex PCR primers (Table 2.1) targeting a unique region in each
group. The PCR experiments were performed using a set of specific primers
and PCR conditions as described by Woodford and co-workers in 2006, the
PCR products were then run on 1% (w/v) agarose gel to study their number
and size in accordance to each phylogenetic CTX-M group (Woodford et al.,
2006). Some of the PCR products were selected to represent the source of
samples from Tripoli and Benghazi, the PCR products were then cut of the gel
and purified using PCR purification kit (QIAGEN GmbH, D-40724 Hilden),
the purified PCR products were sequenced by an automated sequencer (377,
ABI, Perkin-Elmer, CT) using the same amplification primers for each group
of CTX-M family.
The reaction conditions used in the Thermal Cycler were as follows:
94°C for 5min ] 1 cycle

94°C for 25s 1
52°C for 40s | 30 cycles
J
72°C for 50s
63
Table 2.1 Multiplex PCR for CTX-M- groups 1,2,8,9 and 26
CTX-M group DNA sequence Gene size
Group 1
CTX-M-1 F 5 -A A A A A T C A C T G C G C C
A G T T C -3 415
CTX-M-1 R 5-A G C T T A T TC A TC G C C A C G TT
Group 2
CTX-M-2 F 5-C G A C G C T A C C C C T G C T A T T-3
552
CTX-M-2 R 5-C C A G C G T C A G A T T T T T C A G G -3
Group 8
CTX-M-8 F 5-TC G C G T T A A G C G G A T G A T
G C -3
666
CTX-M-8 R 5-A A C C C A C G A T G T G G G T A G C-
Group 9
CTX-M-9 F 5-C A A A G A G A G T G C A A C G G A
TG -3
205
CTX-M-9 R 5-A T T G G A A A G C G T T C A TCA
CC-3
Group 26
CTX-M-26 F 5-G C A C G A TG A C A T T C G G G -3
CTX-M-26 R 5-A A C C C A C G A T G T G G G T A G C- 327
64
2.10.1.2 Detection of A / a c t x - m group 1 and ISEcpl genes
E. coli and K. pneumoniae isolates positive for CTX-M group 1 were
subjected to PCR experiments to examine the incidence of A / a c t x -m - is
encoding genes and the insertion sequence ISEcpl gene that is located
immediately upstream of the P-lactamase gene. Specific primers were
designed to read and amplify the AAzc tx -m group 1 alone and in association
with the ISEcpl (see appendix Table A.2), PCR conditions used were as
follows; 1 cycle of heating at 94°C for 5min followed by 30 cycles of heating
at 94°C for 25s, 52°C for 40s and 72°C for lmin, the reaction ended with 1
cycle of heating at 72°C for 6 min, for amplification of A /a c t x -m group 1. The
same PCR conditions were used to detect AAzctx -m group 1 in association with
ISEcpl with extended annealing time to 90s. Positive controls were not used
as some PCR products were sequenced. When required, new primers were
designed using primer designer version 1.01, scientific and educational
software.
2.10.1.3 Amplification of A /a te m and A /a s h v , A/aAmpC, class 1 integrons
and transposons
K. pneumoniae and E. coli isolates that were confirmed for ESBLs production
were further examined for the occurrence of TEM, SHV, bla^mpc, class 1
integrons transposons Tn402 and Tn27 genes by PCR using specific primers
(see appendix Table A.2). The same conditions applied for A / a c t x -m - is
65
amplification were used in these experiments. The alleles were cut, purified
and sequenced as previously described.
2.10.2 Phenotypic detection of MBLs
Carbapenem susceptibility of the positive isolates to MBLs was performed
using Etest strips (AB BioMerieux, La Plane, France) and the results were
interpreted in accordance with the manufacturer’s guidelines. PCR was also
conducted to study the occurrence of bla$?M-\, blay\u , 6 /« g im , & /« n d m -i, bla\u p ,
bla$\y[.\, blayMM-u & /« A iM -ia n d bla®im - i - The PCR conditions used to amplify
class 1 integron(s) were the same as described in section 2.9.1.1 and for
primers used (see appendix Table A.3) with a slight modification where the
annealing temperature in these conditions was 53°C and the elongation
temperature was 68°C. All the PCR products were run on 1% (w/v) of agarose
gel and the gels were then photographed. The resultant PCR products were
purified from the agarose gel and sequenced using an automated sequencer
(377, AB, Perkin-Elmer, CT).
2.11 Detection of A/aoxA-4sand 1S1999
PCR experiments were performed on K. pneumoniae isolates to detect the
occurrence of Z)/aoxa-48 and the insertion sequence IS1999 using specific
primers targeting the forward and reverse side of both genes (Table 2.2). PCR
products were run in 1% of (w/v) agarose gel in TBE buffer, the
electrophoresed gels were photographed.
66
Table 2.2 Oligonucleotide sequences to detect b la o w - w and IS1999
Primer
Gene target Sequence Reference
name
OXA-48 A 6/aOXA-48 5' TTG GTG GCA TCG ATT ATC GG '3 Poirel, L. et al., 2004
OXA-48 B blaOXA-4S 5' GAG CAC TTC TTT TGT GAT GGC '3 Poirel, L. et al., 2004
IS1999 A 751999 5' CAG CAA TTC TTT CTC CGT G '3 Poirel, L. et al., 2004
IS 1999 B 751999 5 'CAA GCA CAA CAT CAA GCG C '3 Poirel, L. et al., 2004
2.12 Random amplified polymorphic DNA (RAPD) typing
2.12.1 RAPD DNA extraction by Chelex prep
It is a PCR-based technique used to differentiate between bacterial species by
using short primers (Table 2.3) to anneal various locations of the bacterial
DNA. A 24 h growth of K. pneumoniae isolates plated on MacConkey agar
were used without selection and bacterial colonies were picked from the plate
by inserting a sterile 200 pi plastic pipette tip into the colonies and dipped into
50 pi of an autoclaved solution of 5% Chelex® 100 resin (Biorad,
Hertfordshire, UK).
67
To resuspend the mixture in the tube, it was agitated briefly. DNA extraction
was carried out twice by heating the mixture to 89°C for 5 minutes on a heated
block; the samples were immediately transferred to a 4°C chilled block. To
sediment the chelex resin and cell debris, the samples were centrifuged for 5
minutes at 13.000 g and 2 pi of the clear supernatant was used as a template
DNA to run the PCR.
2.12.2 Random amplified polymorphic DNA (RAPD-PCR)
RAPD-PCR fingerprinting was performed on 80 isolates of K. pneumoniae
(12 isolates per reaction) as described by Mahenthiralingam et al., 1996.
RAPD-PCR was conducted using primer 272 (table2.3) for all reactions. For
confirmatory purposes; RAPD-PCR using primer 270 (table 2.3) was carried
out on subsets of K. pneumoniae isolates. PCR master mix was prepared prior
to each experiment (IX PCR buffer, IX Q-solution, 3mM MgCb, 200 pM
dNTPs mixture, 1.6 pM RAPD primer, 1 U of Taq polymerase and 2pl of
Chelex template DNA. PCR was run on a Flexigene Thermal Cycler (Techne
Ltd., Newcastle, UK) using the following PCR conditions; 5 minutes of
heating at 94°C, 4 cycles at 36°C for 5minutes, 72°C for 5 minutes and 94°C
for 5 minutes, followed by 30 cycles of 94°C for 1 minute, 36°C for 1 minute
and 72°C for 2 minutes. The last step was 72°C for 10 minutes.
68
2.12.3 DNA profile analysis by Agilent Bioanalyzer
1 pi of each PCR product was run for 20 minutes on an Agilent Bioanalyzer
2100 (Agilent Technologies UK Limited, Cheshire, UK) and a DNA 7500
chip contained 13 wells was used; 12 wells filled with samples, 1 pi in each
and one filled with the ladder marker. The wells were also loaded with DNA
gel matrix and an internal marker according to the manufacturer’s protocol.
After each run the results were saved as csv files.
2.12.4 GelCompar analysis
All csv-files were converted to a format compatible to GelCompar, similarities
between fingerprints were calculated to the Pearson coefficient and
unweighted pair group method with arithmetic means (UPGMA) was used to
construct the dendrogram.
2.13 Multilocus sequence typing (MLST)
A subset of K. pneumoniae isolates were selected according to the RAPD-PCR
fingerprinting results and MLST analysis was carried out as described by
Diancourt et al., 2005 and the MLST website (http:/ /www .pasteur .fr
/recherche/genopole/PF8/mlst/Kpneumoniae.html) developed by Jolley et al.,
2004. Specific primers were used (Table 2.3) to amplify fragments of the
following 7 housekeeping genes; P-subunit of RNA polymerase (rpoB ),
glyceraldehyde 3-phosphate dehydrogenase (gapA), malate dehydrogenase
69
{mdh), phosphoglucose isomerase (pgi), phosphorine E (phoE), translation
initiation factor 2 (nfB) and periplasmic energy transducer (tonB).
The PCR conditions used for rpoB, mdh, pgi, phoE and nfB were as follows
94°C for 5min ]1 cycle

94°C for 5min -|
50°C for 30s 130 cycles
68°C for lm in ^
68°C for lOmin ]1 cycle
The same PCR conditions were used for gapA and tnoB apart of the annealing
temperature which was 50°C for gap A and 60°C for tnoB. All PCR products
were run on 1% (w/v) Agarose gel and the gels were photographed. All PCR
products were sequenced using an automated sequencer (377, ABI, Perkin-
Elmer, CT) and the same amplification primers apart of in f forward primer
which was replaced with (5’- ACT AAG GTT GCC TCC GGC GAA GC -3’)
and pgi primers were replaced with pgi2F; (5’- CTG CTG GCG CTG ATC
GGC AT -3’) and pgi 2R (5’- TTA TAG CGG TTA ATC AGG CCG T-3’).
70
Table 2.3. Oligonucleotides used for PCR amplification and DNA
sequencing
Gene Prim er
Prim er sequence Reference
target name
ropB ropB F 5 ’-GGCGAAATGGC W GAGAACCA-3 ’ Diancourt et a l., 2005
ropB ropB R 5 ’-GAGTCTTCGAAGTTGTAACC-3 ’ Diancourt et al., 2005
gapA gapA F 5 ’-TG A AA TA TGA CTCC ACTC ACGG-3 ’ Diancourt et al., 2005
gapA gapA R 5 ’-CTTC AGA AG CG G CTTTG A TG GCTT-3 ’ Diancourt et al., 2005
Mdh Mdh F 5 ’-CCCAACTCGCTTCAGGTTCAG-3 ’ Diancourt et al., 2005
Mdh Mdh R 5 ’-CCG TTTTTCCCCA G CA G CA G -3’ Diancourt et al., 2005
Pgi pgi F 5 ’-G AG AA A AA CCTGCCTGTACTGCTGG C-3 ’ Diancourt et al., 2005
P gi Pgi R 5 ’-CGCGCC A CG CTTTATA GCGG TTA AT-3 ’ Diancourt et al., 2005
phoE phoE F 5 ’-ACCTA CCG CA ACA CCG ACTTCTTCG G-3 ’ Diancourt et al., 2005
phoE phoE R 5 ’-TGATC A GAACTGGTAGGTGAT-3 ’ Diancourt et al., 2005
infB infB F 5 ’-CTCG CTG CTG G A CTA TA TTCG -3’ Diancourt et al., 2005
infB infB R 5 ’-CG CTTTCA G CTCA A G A A CTTC-3’ Diancourt et al., 2005
tnoB tnoB F 5 ’-CTTT A T ACCTCG G T ACATC A GG TT-3 ’ Diancourt et al., 2005
tnoB tnoB R 5 ’-ATTCGCCGGCTGRGCRGAGAG-3 ’ Diancourt et al., 2005
Integrase
VAF 5 ’ GCCTGTTCG G TTCGTA AG CT 3 ’
gene
PAPD-
M ahenthiralingam et al.,
PCR 272 5 ’- AGC GGG CCA A -3 ’
1996
Primer
PAPD-
M ahenthiralingam et al.,
PCR 270 5 ’- TGC GCG C G G G - 3 ’
1996
Primer
71
2.14 Plasmid identification
Plasmids are circular extra-chromosomal DNA; they are known to play a role
in changing the diversity of the bacterial genome by acquiring or losing genes
such as antibiotic resistance genes, subsequently contribute to the movement
and transfer of resistance mechanisms from bacteria to bacteria by means of
horizontal gene transfer (Carattoli, A. et al., 2005). PCR-based replicons
typing was performed to identify plasmids contributed to the dissemination of
ESBL and MBL genes among Libyan isolates using 5 multiplex and 3 simplex
PCR experiments as described by Carattoli, A. et al., 2005. This procedure is
used to identify the major plasmids that are known as incompatible plasmids
by recognizing FIA, FIB, FIC, HI1, HI2, II, Iy, L/M, N, P, W, T, A/C, K, B/O,
X, Y, F and FIIA using eighteen pairs of primers designed to be conducted on
8 PCRs. The 5 multiplex PCRs are designed to recognize three plasmids for
each reaction (see appendix Table A.4). Positive controls were used to
comaper size of plasmids. The PCR conditions used to detect all plasmids
apart of F simplex were as follows

94°C for lmin 1
60°C for 30s | 30 cycles
J
72°C for lmin
72°C for 5min 11 cycle
72
Whereas the conditions of F simplex PCR were almost the same with only one
difference as the annealing temperature was changed to 52°C.
2.15 Transconjugation experiments
Conjugation experiments were carried using E. coli J53 and GFP as recipients.
Fresh colonies of parents and recipients were grown separately on LB broth
media (Fisher Scientific, USA Products) in 50 ml Falcon tubes and incubated
overnight at 37°C for 18 h. Each isolate of parents was mated with E. coli J53
or GFP E. coli in aliquots of 1:1 in a fresh LB broth media and incubated
overnight at 37°C in shaking incubator. Transconjugants were selected by
culturing 100 pi of each mating mixture on LB medium (Fisher Scientific,
USA Products) supplemented with 200 pl/ml of sodium azide and 10 mg/1 of
ceftazidime. Parents that were mated with GFP E. coli, the selection was
performed on L.B agar supplemented with 50mg/l of rifampicin and 10mg/l of
ceftazidime. The plates were subsequently incubated overnight at 37°C for 18
h. Pure colonies of E. coli from each plate were picked and transferred to a
fresh LB broth media supplemented with 200 pl/ml of sodium azide and 10
mg/1 of ceftazidime for E. coli J53 transconjugants and with 50mg/l of
rifampicin and 10mg/l of ceftazidime for GFP E. coli transconjugants. The
transconjugants were then plated on LB media supplemented with the same
concentrations of antibiotics used for parents and incubated overnight at 37°C
for 18 h. The transconjugants that were grown on LB media were stored at -80
°C for further investigation. PCR experiments were performed on
73
transconjugants targeting blacix-u group 1 encoding genes and ISEcpl for K.
pneumoniae and E. coli using the forward and reverse primers from table (1).
2.16 Southern hybridisation
2.16.1 Characterization of chromosomally and plasmid mediated
resistance genes.
2.16.1.1 Preparation of plugs of whole genomic bacteria DNA
Whole genomic DNA o f the bacteria was used to prepare plugs to detect
chromosomally and plasmid mediated genes. Bacterial cultures were grown
overnight at 37°C. One loop of the fresh colonies of each isolate was
suspended in 3 ml of normal saline and the optical density 600 (OD 600) of each
isolate was measured and the formula (1.5/measured OD Multiplied by 300) to
adjust the volume of cells to the equivalent of 300 pi in accordance to the
OD6oo- The suspended cells were then centrifuged at 13 Kg using mini
centrifuge (Minispin centrifuge, Hamburg, Germany) for 30s and the
supernatant removed. Cells were then re-suspended in 300 pi of normal saline
and transferred to a 50°C block heater. Cells were lysed by adding 2-3 drops of
25 mg/ml of lysozyme and a 2.5 % (w/v) of pre-warmed (50°C) low melting
point agarose was quickly pipetted and gently mixed and quickly dispensed
into PFGE plugs components and dried at room temperature for 30 min. 5
plugs of each set were then transferred into a 24 well plate and 2 ml of lysis
buffer (10 mM Tris-Hcl, pH 7.2, 50 mM NaCl, 0.2% sodium deoxycholate,
0.5% N-Lauroylsarcosine) was added and supplemented with 80 pi of 25mg/l
74
of lysozyme. The plugs were then incubated at 37 °C for 1.5 hrs. The plugs at
this stage were washed with 2 mis of IX TE buffer (10 mM Tris-HCl, 50 mM
EDTA pH 8.0; Bio-Rad) at 37°C for 30 mins. The TE was replaced with 2
mis of proteolysis buffer (100 mM EDTA pH 8.0, 0.2% sodium deoxycholate,
1% and N-Lauroylsarcosine; Bio-Rad) 20 pi of 10 mg/1 of Proteinase K and
incubated at 50°C for 18 hrs. After the proteolysis buffer was removed, the
plugs were then washed five times with IX TE buffer in shaking incubator at
37°C for 30 mins.
One plug of each set was transferred to a new 24 well plate and washed with
0.1 X TE buffer at 37 °C for 30 mins. The plugs were then washed twice with
2X SI buffer at room temperature for 15 mins each. The 2X SI buffer was
removed and replaced with IX SI buffer and washing was performed at room
temperature for 15 min. The SI buffer was then removed and 1 pi of 20U of
Si endonuclease (Promega, USA) was added and the plugs were incubated at
37°C for 45 min. 100 pi of ES buffer (0.5 M EDTA, pH 8; 1% N-
Lauroylsarcosine) was added to stop the digestion. PFGE gels were prepared;
0.88% (w/v) agarose in 0.5 X of TBE buffer (45mM Tris-base, 45 mM boric
acid, 1 mM EDTA, pH 8.0; Bio-Rad) and 20 pi of ethidium bromide was
added to stain the gels. Plugs were loaded into the gels and the gels were run
in the PFGE tank (CHEF-DRIII system, Bio-Rad laboratories). Migration of
DNA was performed at 9°C with initial switch time of 5 and final switch time
of 45 for 20 hrs at 6 volts and 120 0 angle. Lambda Ladder was used as a DNA
size marker.
75
2.16.2 Pulsed Field Gel Electrophoresis (PFGE) Typing
Plugs of the whole genomic DNA of the target bacterium was prepared as for
Spe 1 digests described by Patzer & Dzierzanowska, 2007. Each plug was
washed with 0.01 x of TE buffer shaking at 37°C for 30 min, followed by
washing twice with 300 pi of 2x of Xbal fast digestive buffer (Fermentas,
Sheriff Hutton Industrial Park, York, UK) for 15 min at room temperature and
once with 300 pi lx of Xbal fast digestive buffer for 15 min. The DNA in
plugs was then digested with 3.5 pi of Xbal overnight at 37°C for K.
pneumoniae and E. coli. The same steps were performed on plugs made of
whole genomic DNA from P. aeruginosa but washed with Spel buffer and
digested with 1 pi of Spel enzyme. Separation of Xbal and Spel digested
DNA was performed by using PFGE apparatus (CHEF-DRIII system, Bio-Rad
laboratories) and DNA migration was conducted using the following
conditions; initial switch time at 5s and final switch time at 45s, 6V/cm and
120° angle for 20h with cooling at 9°C, using TBE buffer (0.5x Tris borate,
0.5mM EDTA), Lambda ladder DNA was used as a marker to size DNA. The
interpretation of similarities between bacterial species was performed as
described by (Tenover et al., 1995). The resultant PFGE Gels were
photographed and dried overnight on a Whatman filter paper (15 cm * 15 cm)
blotting paper, the gels were then re-hydrated, denatured using a denaturing
buffer (0.5M NaOH, 1.5M NaCl) for 30 min at room temperature, neutralized
using a neutralizing solution (0.5M Tris-HCl, pH 7.5, 1.5M NaCl) for 30 min
76
at room temperature. The gels were then transferred to a hybridization tube
contains pre-hybridization solution at 65 °C and probed with a P radio
labelled CTX-M-15 template DNA and CTX-M-15/ISEcpl for K. pneumoniae

32 •
and E. coli and the P. aeruginosa PFGE gels were probed with a P radio
labelled VIM-2 as described by Patzer et al., 2009.
2.16.3 Colony Blotting.
Colony blotting experiments were carried out by using a modification of the
procedure of (Ivanov & Gigova, 1989). MacConkey agar plates were spotted
with the isolates of interest and incubated overnight at 37°C for 18 h.
MacConkey agar plates with bacterial isolates were photographed using digital
camera and then overlaid with a circular membrane (Hybond™ , Amersham
Pharmacia, UK), for at least 2 min, so the bacterial isolates will have been
transferred to it. The membranes were then removed by a sterile forceps and
placed colony side up on a presoaked 15cm Whatman blotting paper
(Whatman inc. Sigma-Aldrich, Sanford, UK) with 5% of SDS (sodium
dodicyl sulphate) for 5 min at room temperature. The membranes were then
carefully transferred to a 15cm Whatman blotting paper to remove any

•y
excess moisture, the membranes were then placed colony side up on 15cm
Whatman blotting paper presoaked with denaturing solution (1.5 NaCl, 0.5 M
NaOH) for 5 min.
The membranes were then carefully removed and dabbed dry on 15cm2
Whatman blotting paper and transferred and floated colony side up in
77
neutralizing solution (157 g. Tris-HCl, 174 g. NaCl in 2L of H 20 pH 7.5) for
5 min. The cellular debris was then carefully removed and washed with 6X
SSC (6 ml of 20X SSC in 20 mis of demonized water) and dabbed dry. The
membranes were then dried at 80°C for at least 3h to fix the DNA to the
membrane filters. The membrane filters were then transferred to hybridization
tube provided with hybridization solution (6X SSC, 0.1 % (W/V)
polyvinylpyrrolidine (PVP), 1 ml of 0.5 % (W/V) SDS, 400 pi of 0.1% (W/V)
ficoll, 400 pi of Milk and 300 pi of 150 pg/ml'1 denatured spermatozoid
DNA). The hybridization tube was then incubated at 65°C prior to probing
with gene of interest.
2.16.4 In gel hybridization
The resultant PFGE gels were photographed and dried at 50°C for 18 hrs, the
gels were then hybridized as follows; rehydrated in DNA free water for 30
mins at room temperature, the DNA in gel was denatured for 30 mins using
denaturing solution (NaCl, 0.5 M NaOH) and neutralized by neutralizing
solution (Tris-HCl, NaCl) for 30 mins. The gels were then transferred to
hybridization tubes with pre-hybridization solution (6X SSC, 0.1% (W/V)
polyvinylpyrrolidine (PVP), 1 ml of 0.5% (W/V) SDS, 400 pi of 0.1% (W/V)
ficoll, 400 pi of Milk and 300 pi of 150 pg/ml’1 denatured spermatozoid
DNA) and incubated at 65°C overnight. The hybridized gels were
subsequently probed. Gels were then washed twice, once with 2X SSC
(Sodium Citrate), 0.1% (W/V) SDS and once with 0.1 X SSC, 0.1% (W/V)
78
SDS. The gels were then wraped in cling film and transferred to a cassette and
a Hyperfilm™ (Amersham, GE Healthcare, Life Sciences) was firmly pressed
on the gel and frozen at -80°C for 18 hrs. Developer and fixer were used to
detect the appearance of any radio labeled spot on the Hyperfilm.
2.16.5 Labelling DNA Probes
To produce high specific activity probes, labeled DNA was generated using
random oligonucleotides, and anneal to specific sites on the DNA template.
The Klenow will use the primer-template complex as a substrate and
synthesize a new DNA by incorporating monophosphates at the free 3'-OH
group. Radio-labeling is performed by exchanging the nonradioactive with the
radioactive in the reaction mixture. The radio-labeled gene will then serve as a
sensitive hybridization probe, it is used in southern and northern blots and in
Situ hybridization techniques. The genes of interest (&/<2c t x -m - i 5 alone and in
association with ISEcpl, blayim -2, tniC, blajû, blasnv, ISCR2 and 6/# t m b - i )
were amplified by PCR using specific primers targeting the forward and
reverse regions of the gene to be used as a template DNA to probe the
hybridized membranes. 15 pi of the template DNA was mixed with 8 pi of
DNA free water and 10 pi of random 9-mer primers (Agilent Technologies -
Stratagene - USA Products) were added in a screw capped Eppendorf tube.
The mixture was firstly boiled in a water bath for 5 min and immediately 10 pi
of 5X dCTP buffer, 2,5 pi of the radioactive phosphorus 32P and 1 pi of Exo(-)
Klenow (Agilent Technologies - Stratagene - USA Products) were added to
79
the mixture and transferred to a jar made of lead and incubated at 37°C for 15
min to allow the production of the radio-labeled template DNA. The radio-
labeled product was then pipetted into a silica gel column (Nick™ columns
Sephadix, G-50 DNA Grade, illustra, GE Healthcare, Life Science, UK). The
column was then washed with 320 pi of washing buffer (0.1 M Tris-Hcl
Buffer, PH 7.5) followed with 430 pi of the same washing buffer to an
Eppendorf tube to elute the radio-labelled gene purified. The radio-labeled
PCR product was then boiled in a water bath for 6 min to denature the double
stranded template DNA, the probe was then added to the previously incubated
membranes or gels (see sections 2.15.3 and 2.15.4) in the hybridization tube
and incubated over night at 65 °C.
2.17 Cloning Experiments
Cloning experiments were performed on an A. xylosoxidans isolate trying to
obtain the full sequence of the new MBL gene. The cloning experiments were
carried out by chemical transformation (Blue/White) screening test by using
the plasmid vector (pCR®4-TOPO®) and E. coli 5DHa supplied by TOPOIO
cloning kit supplied by (Invitrogen Ltd, Inchinnan Business Park, 3 Fountain
Drive, Paisley, UK). The 3kb PCR products were amplified from the A.
xylosoxidans and purified before using it in the cloning experiments. TOPO
cloning reaction was performed by mixing the 3kb class 1 integron, salt
solution (1.2 M NaCl 0.06 M MgC12) and TOPO vector at room temperature
and then kept on ice. To perform transformation, 2pi of the reaction was then
transferred into a vial containing chemically-competent E. coli and incubated
80
on ice for 30 minutes. The E. coli was heat shocked at 42°C for 30s without
shaking and immediately returned to ice and 250 pi of S.O.C broth medium
then added and incubated at 37°C for lh. A total of 50pl of the broth culture
was streaked on L.B. Agar (Fisher Scientific, USA products) plates
supplemented with 50 mg/1 of kanamycin, X-galactose and isopropyl-|3-D-
thiogalactoside (IPTG) and then incubated at 37°C for 18h. The white colonies
were picked up and grown overnight in L.B broth, the TOPO vector was then
extracted from the cells by miniprep kit and sequenced using the primers M l3
forward and reverse.
2.18 Purification of TMB-1
2.18.1 Expression
TMB-1 was purified directly from the A. xylosoxidans isolate grown
overnight at 37°C in flasks containing 4x 50ml of Terrific broth (Sigma, St.
Louis, MO, USA) supplemented with 50mcg/ml of kanamycin, the cultures
were then incubated shaking at 37°C. Each flask was inoculated with 4x 1L of
Terrific broth with 50ug/ml of kanamycin and flasks incubated at 37°C and
225 rpm. The production of the protein was induced by IPTG (final
concentration 0.1 mM) when O.D 6oois between 0.6-0.7. Cells were centrifuged
at 7000 g for 10 min at 4°C. The expression of protein was confirmed using
Sodium SDS-Page.
81
2.18.2 Periplasm isolation
To perform large scale protein preparations of periplasmic cellular extracts, it
was necessary to treat cells with lysozyme. The methods used were that of
Avison et al., 2011 and Samuelsen et al., 2008. The cell pellets were
resuspended in buffer (50mM Tris-HCl, lOOuM ZnC12, 0.02% NaN3 pH 7.2).
The lysozyme was then added to a concentration of 200pg/ml. The suspension
was then incubated rotating at room temperature for 15-20 min. CaC12 was
then added to a concentration of lOmM, the suspension was then centrifuged
at 9000xg or 18000 rpm for 20 min. at 4°C.
2.18.3 Purification of (3-lactamase from crude periplasmic cell extract
The crude cell extract was loaded on to 50 ml Q-Sepharose column (Q-
Sepharose HP column, Pharmacia, GE Healthcare, UK) that was previously
pre-equilibrated with 100 ml of buffer (buffer (50mM Tris-HCl, lOOuM
ZnC12, 0.02% NaN3 pH 7.2).The protein was then loaded and eluted using
400 ml NaCl gradient. The eluted fractions were collected and checked for p-
lactamase activity using Nitrocefin. Purity of fractions that showed p-
lactamase was performed on SDS-PAGE (2-14% NuPAGE Bis-Tris mini
gels).
82
2.18.4 Gel-filtration
Column was pre-equilibrated with two column volume of washing buffer (see
section 2.18.2), the protein was loaded through a super loop (flow lml/min)
and then wash or elute the protein with (100-300) of washing buffer. Fraction
were then collected and checked for P-lactamase using Nitrocefin, the active
fractions were run on SDS-PAGE and stored at 4°C. TMB-1 was analysed
using nitrocefin +/- EDTA and SDS-PAGE. TMB-1 was concentrated to
1.94mg/ml.
2.19 Kinetics assay:
Steady-state kinetics was performed at 25°C in a spectrophotometer
(SpectramaxPlus, Molecular Devices) using 96 well plates (BD Falcon UV
microplates, BD Biosciences, USA) (Samuelsen et al., 2008). All substrates
(ceftazidime, cefoxitin, cefuroxime, piperacillin, ampicillin, imipenem,
meropenem and ertapenem) were tested as duplicates using 50mM HEPES pH
7.2, lOOpM ZnCC, 0.02% NaN 3 , and 0.1 mg/ml bovine serum albumin
(Sigma-Aldrich) as a buffer system. The kinetic data were analysed by non
linear regression (GraphPad Software, San Diego, CA).
83
Chapter Three
Characterization of Multi-drug
resistant Klebsiella pneumoniae from
Tripoli & Benghazi, Libya
3.1 Introduction
K. pneumoniae can be isolated from a variety of different sites, locations and
environments such as, water and soil, or from hospitalised patients or from
animals. Such variation in habitats provides K. pneumoniae with the
opportunity to spread quickly and as a consequence it can cause infections
(Podschun & Ullmann, 1998). Infections due to MDR strains of K.
pneumoniae have been reported world-wide in neonatal wards, ICUs,
paediatric hospitals (Podschun & Ullmann, 1998; Bagattini et al., 2006), UTIs
and lower respiratory tract infections (Gori et al., 1996; Podschun & Ullmann,
1998; Cartelle et al., 2004; Valverde et al., 2008; Kiratisin, 2008). It is
increasingly reported year on year (Grobner et al., 2009; Lim et al., 2009) and
thus represents a major clinical threat particularly for immunocompromised
patients (Oteobe^ al., 2009).
The frequent use of extended-spectrum cephalosporins, particularly in ICUs is
considered a leading factors contributing to epidemic and endemic outbreaks
of nosocomial infection as a result of the emergence of MDR Gram-negative
pathogens producing ESBLs (Gori et al., 1996;Valverde et al., 2008). ESBLs
are the most prevalent enzymes produced by multi-resistant strains of K.
pneumoniae and are capable of hydrolysing most p-lactams particularly third
and fourth generations cephalosporins (Wei et al., 2005).
85
Nosocomial infections caused by ESBLs producing K. pneumoniae have
become a major problem in the United States, Europe, Asia (Livermore,
2009), Africa (Gori et al., 1996), Brazil and Spain (Rodriguez-Bano et al.,
2010). ESBLs are often carried on plasmids of different sizes and types
reflecting the frequency and epidemiology of these enzymes (Gori et al.,
1996). CTX-M-type ESBLs are encoded by genes carried on plasmids of
different types such as; IncFl, IncFII, IncH12 and Incl which are classified as
narrow host-range types of plasmids and known to mobilize blacrx-u -\5 and
ISEcpl. Furthermore, IncN, IncP-l-a, IncL,/M as well as Inc A/C are broad
host-range plasmids and effective as transmissible elements and play
important roles in the dissemination of ^/actx-m-is genes (Pitout, 2010;
Carattoli, 2009). Such replicons can act as major vehicles for the horizontal
transfer o f genes responsible for antibiotic resistance that cause CAIs and
HAIs (Colinon et al., 2007). CTX-M type extended spectrum-(3-lactamases are
considered the most prevalent ESBLs among E. coli and K. pneumoniae.
These enzymes have originally been derived from chromosomal P-lactamase
from Kluyvera spp. (Dedeic-Ljubovic et al., 2010).
blacix-u -\5 is one of the most important enzymes of the 120 variants of CTX-
M type ESBLs found to date (http://www.lahey.0 rg/Studies/0 ther.asp#tablel)
and was first discovered in India, France and Japan in the 1980s and recently
worldwide (Yu et al., 2004; Lartigue et al., 2007; Touati et al., 2006; Abbassi
et al., 2008; Gonullu et al., 2008; Walsh, 2006). blac\x-u -\5 has a broader
86
substrate profile than many other CTX-Ms due to mutations around the active
site (Pitout, 2010). Several reports have mentioned the occurrence of blacxx-u-
15 associated with the insertion sequence ISEcpl located upstream of the CTX-
M gene in E. coli and K. pneumoniae from Nigeria, Norway, Tunisia, UK and
France (Touati et al., 2006; Abbassi et al., 2008; Eckert et al., 2006; Kiratisin
et al., 2008; Ben Salma et al., 2011; Younes et al., 2011).
blacjx-M-\5 has also been detected in the Mediterranean area, the Middle East
and the Arab Gulf region. The CTX-M-15 gene has been found in clinical
isolates of E. coli from Cairo, Egypt and associated with the insertion
sequence, IS Ecpl (Khalaf et al., 2008). K. pneumoniae and E. coli harbouring
blacix-u -\5 were found disseminated in neonatal wards and ICUs in Saudi
Arabia (Al-agamy et al., 2009), Algeria (Ramadani-Bouguessa et al., 2006)
and Kuwait (Dashti et al., 2010). Similarly, blacix-M-is was found plasmid
mediated in clinical isolates of E. coli collected from Egypt (Mohamed Al-
Agamy et al., 2006).
This chapter describes the emergence of MDR K. pneumoniae isolates from
clinical settings (patients and hospital environment) and non-hospital
environmental isolates. The phenotypic characteristics and the antibiotic
resistance profile of 80 K. pneumoniae isolates are determined and discussed.
87
3.2 Results
3.2.1 Antimicrobial susceptibility testing
K. pneumoniae isolates collected are listed in table 3.1. The MIC 50 , MIC9 0 and
MIC ranges of 80 isolates of clinical, hospital environment and non-hospital
environment K. pneumoniae are presented in table 3.2. These results show that
MIC 50 and MIC9 0 of ceftazidime was higher that of cefotaxime. The highest
MIC 50 and MIC9 0 were observed for piperacillin/tazobactam whereas the
lowest was for the carbapenems; imipenem and meropenem. The highest level
of resistance (95 %) has been observed against the antibiotics piperacillin and
ampicillin. Thirty five out of eighty (43.75 %) of K. pneumoniae exhibited

resistance against piperacillin/tazobactam. The results also showed that 52/80
(65%) showed resistance to amoxicillin/clavulanic acid combinations and 3
others showed intermediate resistance to amoxicillin/clavulanic acid
combinations. Resistance to aminoglycosides varied; 2/80 (2.5 %) showed
resistance to amikacin, whereas 49/80 (61 %) were resistant to gentamicin.
33/80 (41 %) were resistant to ciprofloxacin and another one was intermediate.
Fifty six out of 80 isolates (70 %) were resistant to cefuroxime whereas 48/80
(60 %) and 49/80 (61 %) displayed resistance to ceftazidime and cefotaxime,
respectively; and 46/80 (57.5 %) were indicated by Phoenix as ESBL positive.
Of those that are ESBL positive, resistance was observed for
amoxicillin/clavulanate and piperacillin/tazobactam with 38/46 (82.6 %) and
29/46 (63%), respectively.
88
3.2.2 Genotypic detection of 6 / « o x a -4 8 and ESBLs
3.2.2.1 The prevalence of CTX-M groups 1, 2, 8, 9 and 26
The results of detection of the occurrence of CTX-M groups 1, 2, 8, 9 and 26
are shown in Figure 3.1. This experiment was based on the amplification of
part of the targeted gene of each group of CTX-M-type ESBLs. 50/80 (62.5
%) o f the K. pneumoniae isolates demonstrated the presence of CTX-M group
1. None of the isolates produced any PCR products when specific primers
were used for CTX-M groups 2, 8, 9 and 26.
Table3.1 Dissemination of K. pneumoniae in Tripoli and Benghazi, RAPD clusters,

MLST and blagx-w group 1 results
Number of RAPD WaCTX-M
Location isolates clusters MLST group 1
A l-Jam horiya hospital Benghazi n=21 1,2 & 5 ST147, ST101 16
Al-Jala hospital o f Benghazi n=8 1,2 & 6 ST101 5
7th o f O ctober hospital n=8 2 ST15 (n=2) 7
Kwaifia hospital Benghazi n=5 1,2 & 6 ST29 2

Benghazi Pediatric hospital n=6 1& 2 0 5
Tripoli medical centre n=l 5 0 0

Tripoli M ilatiry hosital n=2 1, 2 & 4 0 1
Tripoli maternity hosital n=l 1 1& 2 ST70 6
Burn and plastic surgery centre o f Tripoli n=12 1,2,4 & 6 ST111, ST15 7
Tripoli pediatric hospital n=l 1 0 0

Benghazi lake n=l 1 0 0
Syria area Benghazi n=2 6 ST506, ST486 1
Keesh area Bemghazi n=l 6 0 0
D ollar area Benghazi n=l 2 ST511 1
89
Table 3.2 MIC50 and MIC90 of K. pneumoniae
Antibiotic MIC 50 MIC90 Range mg/1
Ceftazidime 16 32 4-32
Cefotaxime 8 64 2-64
Imipenem 0.5 1 0.125-1
Meropenem 0.5 1 0 .1 2 5 -1
Aztreonam 16 32 8-32
Piperacillin/Tazobactam 32 128 4-128
Ciprofloxacin 4 8 0.5-8
Ampicillin 16 64 4-64
Gentamicin 8 16 2-16
3.2.2.2 Detection of CTX-M-15 genes and ISEcpl
The results of detection of the incidence of blact x - m -15 in a subset of 11 K.
pneumoniae isolates (AES64, AES 178, AES261, AES268, AES273, AES274,
AES280, AES970, AES973, AES984 and AES 1001) are shown in Figure 3.2.
Sequencing of some PCR products showed the occurrence of 6/< z c t x -m - i 5 genes
in Libyan K. pneumoniae (B.18- B31). The results suggest that 6 / a c t x -m - i 5 is
the gene responsible for the production of ESBL and mediates extended
spectrum cephalosporin resistance in some Libyan K. pneumoniae.
Amplification of blacjx-u-\s genes in association with the insertion sequence
90
ISEcpl are illustrated in Figure 3.3. In addition Figure 3.4 show the
association between blacix-ugroup 1 gene with the insertion sequence ISEcpl

and the differences that are noticed due to the presence/absence of an intact
copy of the insertion sequence in some of these isolates. The insertion
sequence ISEcpl is the promoter for the movement and expression of the
cefotaximase encoding gene and it is more often than not located upstream of
the p-lactamase gene (Poirel et al., 2003). PCR products obtained by
amplification of blaax-u -\5 genes and ISEcpl from 11 of K. pneumoniae

isolates using two forward primers (ISEcpul and ISEcpu2) targeting two
different sites on the insertion sequence) and the standard reverse primer
(CTX-M-15 R) produced different sized products.
The results suggest the occurrence of a deletion event in ISEcpl in some of

the isolates, and PCR using different primers failed to amplify the insertion
sequence and Wuctx-m-is and consequently appeared negative (Figure 3.4).
The deletion was confirmed by using the forward primer ISEcup2 with the
reverse primer for the blacjx-M-is gene. On the same isolates, the results of the
amplification of blact x - m - i s gene and ISEcpl using ISEcupl forward primer
with CTX-M-15 reverse primer were able to prove the occurrence of blacjx-M-
15 in association with partial copy of ISEcpl (Figures 3.3 & 3.5).
91
m
400 bp
200 bp
Figure 3.1 Multiplex PCR experiment to detect the incidence of CTX-M

type ESBLs groups 1, 2, 8 , 9 and 26. Lanel: Marker. Lane2: K.
pneumoniae isolate AES7. Lane3: AES 8 . Lane4: AES48. Lane5: AES53.
Lane6 : AES59. Lane7: AES64. Lane8 : AES6 6 . Lane9: AES67. Lane 10:
AES6 8 . Lanel 1: AES73. Lanel2: AES74. Lanel3: AES85. Lanel4:
AES 103. Lanel5: AES104. Lanel 6 : AES135. Lanel7: AES136. Lanel 8 :
AES140. Lanel9: AES141. Lane20: AES145.
400 bp
200 bp
Figure 3.2 PCR experiment to detect the incidence of blact x - m - i s in K.

pneumoniae isolates. Lanel: Marker. Lane2: AES64. Lane3: AES178.
Lane4: AES261. Lane5: AES268. Lane6 : AES273. Lane7: AES274.
Lane8 : AES280. Lane9: AES970. Lanel0: AES973. Lanel 1: AES984.
Lanel2: AES1001. Lanel3: Negative control. Lanel4: Marker
92
400 bp
200 bp
Figure 3.3 PCR experiment to detect the incidence of blact x - m group 1 in

association with ISEcpl in K. pneumoniae isolates. Lanel: AES64.
Lane2: AES178. Lane3: AES261. Lane4: AES268. Lane5: AES273.
Lane6: AES274. Lane7: AES280. Lane8: AES970. Lane9: AES973.
LanelO: AES984. Lanel 1: AES1001. Lanel2: Negative control. Lanel3:
Marker
100 bp
ZOO bp
Figure 3.4 PCR experiment to detect disrupted ISEcpl sequence in K.

pneumoniae isolates. Lanel: AES64. Lane2: AES178. Lane3:
AES261. Lane4: AES268. Lane5: AES273. Lane6: AES274. Lane7:
AES280. Lane8: AES970. Lane9: AES973. LanelO: AES984.
Lanel 1: AES 1001. Lane 12: Negative control. Lanel3: Marker.
93
ISEcpu2 ISEcpul
ISEcpl A/acTX-M-i5
Figure 3.5 Diagram showing the genetic environment of & /# c t x -m -15

encoding gene and the insertion sequence ISEcpl located upstream
of the cefotaxime resistance gene. Arrows of ISEcpul (Ho et al.,
2005) & ISEcpu2 (Leflon-Guibout et a\., 2004) indicates the target
of each primer
3.2.2.3 Detection of TEM & SHV in K. pneumoniae isolates
Blotting of 80 K. pneumoniae isolates with TEM and SHV genes are presented
in Figures 3.6A, 3.6B, 3.7A, 3.7B, 3.8A, 3.8B, 3.9A & 3.9B and the results
are summarised in Table 3.3. These results showed that 52 (65%) isolates of
K. pneumoniae were positive for blasuv genes and 27 (33.7%) were positive
for blajem genes. The occurrence of 6 / « c t x -m i 5, blasuv and blajem genes
together was detected in 12 isolates, whereas 16 isolates showed the both
presence of blasuv and blajEu- The results also showed that blasuv genes were
mostly detected in clinical settings (51.25%) compared to those K.
pneumoniae isolates found in the hospital environment (10%). A low
percentage o f SHV genes were observed in environmental isolates collected
outside the hospital. However, the incidence of blajEM among Libyan K.
94
pneumoniae in this study was 26.3% in clinical isolates and 7.5% in the
hospital environment.
Table 3.3 The incidence of blacrx-u group 1, Tn402, blajEM & blaSnv
encoding genes and mobile genetic elements ISCR2 in Libyan K. pneumoniae
isolates
Clinical isolates Hospital environmental Environmental Total %
isolates isolates
CTX-M group 1 40 (n=80) 10 1 68.75%
Tn 402 19 (n=80) 1 2 27.5%
SHV 41 (n=80) 8 3 65%
TEM 21 (n=80) 6 0 33.75%
ISCR1 13 (n=80) 3 1 21.25%
Figure 3.6 Blotting of AT. pneumoniae isolates (1-47) and probing with b la TEU.
A. MacConkey Agar plate. B. Blotting and probing with radio-labelled Motem

of plate A.
95
A B
Figure 3.7 Blotting of K. pneumoniae isolates (48-80) and probing with

blajEM A. MacConkey Agar plate. B. Blotting and probing with radio
labelled blajEM of plate A.
Figure 3.8 Blotting o f K. pneumoniae isolates (1-47) and probing with blasuv.
A. MacConkey Agar plate. B. Blotting and probing with radio-labelled blasuv
of plate A.
96
A B
Figure 3.9 Blotting of K. pneumoniae isolates (48-80) and probing with

blasHx. A. MacConkey Agar plate. B. Blotting and probing with radio-labelled
WasHvgene of plate A.
3.2.2.4 CTX-M group 1 type ESBLs

Blotting and probing of 80 K. pneumoniae isolates with blacix-u - \ 5 template
DNA, labelled with radioactive phosphorus 32P, are summarised in Table 3.3
and illustrated in Figures 3.10A, 3.10B, 3.11A & 3.11B. 51/80 (63.8 %) were
positive for blacrx-u group 1 and 40 out of those 51 (78.4%) were isolated
from blood, urine, pus, sputum, burn ward and sepsis samples collected from
patients in different hospitals in Tripoli and Benghazi. The presence of blact x -
m group 1 positive K. pneumoniae in the hospital environments was 10/51
(19.6%) and reflects the incidence and prevalence of blacrx-u group 1 in
Libyan hospitals. Thus, in total 50/51 blacvx-u group 1 positive K.
pneumoniae were from patients or the hospital environment and is very high
compared to the spread of blacix-u group 1 genes in the community and
97
environment outside the hospitals. Only one isolate of K. pneumoniae
collected from Benghazi streets was found carrying blacrx-u group 1 genes.
Figure 3.10 Blotting and probing of K. pneumoniae isolates (1-47)

with blacrx-u-i 5• A: MacConkey Agar culture. B: Blotting and
probing with radio-labelled blacrx-u-xs amplicon of plate A.
Figure 3.11 Blotting and probing of K. pneumoniae isolates (48-

80) with blacrx-u-i5 • A: MacConkey Agar cultures. B: Blotting and
probing with radio-labelled blacrx-u-\5 amplicon of plate A.
98
3.2.2.5 Detection of A/aoxa-4 8 and IS1999
PCR experiments on K. pneumoniae failed to amplify 6/<2 o x a -48 and IS 1999.
3.2.3 Characterisation of plasmids carrying A / a c t x - m group 1/lSEcpl
Figure 3.12 shows S 1 endonuclease digestion followed by PFGE of genomic
DNA separating chromosomal DNA from plasmids in 14/28 selected K.
pneumoniae isolates (#AES8, AES48, AES135, AES140, AES141, AES216,
AES274, AES275, AES279, AES280, AES281, AES506, AES722, AES808b,
AES809E). An additional figure showing the same application with the other
14/28 K. pneumoniae isolates (AES809, AES817, AES203, AES836,
AES939, AES961, AES942, AES188, AES994, AES960, AES970, AES975,
AES977 & AES982) is presented in Appendix B.6. The selection criterion was
based on prevalence of clinical samples but also included hospital
environmental isolates and the single K. pneumoniae found in the streets. This
experiment was undertaken to examine the incidence of plasmid mediated
blacjx-u group 1 and ISE cpl genes. Probing of the PFGE gel from Figure 3.12
with radio-labelled blacix-u group I/ISEcpl is shown in Figure 3.13. Probing
of the PFGE gel from figure B.6 is presented in Appendix B (Figure B.7).
These results clearly demonstrated that blacix-u groupl/ISEc/?/ has been
detected on plasmids in 14 isolates of K. pneumoniae on seven different
plasmid sizes - 50, 75, 100, 150, 275, 300 and 425kb. Four isolates (AES8,
AES135, AES140 & AES141) carry blacxx-u groupl/IS£cp/ on plasmids of
300kb, 3 isolates (AES506, AES970 & AES982) carry blac tx -m
99
group 1/lSEcpl on the same size of plasmids (175kb), whereas 3 isolates
(AES274, AES280 & AES281) carrying blacjx-MgroupIflSEcpl on a 75kb
plasmid, blactx - m group 1/ISjEc/?/ were found on a lOOkb plasmid in K.

pneumoniae isolate AES275 and in the hospital environmental isolate,
AES722, on a plasmid of 50kb, and on a plasmid of 275kb in a clinical isolate,
AES48, that was cultured from a blood sample. The K. pneumoniae isolate,
AES817, found in on the Benghazi streets carry blacix-ugroup 1/lSEcpl on a
425kb plasmid.
400 kb
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 3.12 PFGE of SI digests for K. pneumoniae AES

isolates. Lanel: Marker. Lane2: AES8. Lane3: AES48. Lane4:
AES135. Lane5: AES140. Lane6: AES141. Lane7: AES216.
Lane8: AES274. Lane9: AES275. LanelO: AES279. Lanel 1:
AES280. Lane 12: AES281. Lanel3: AES506. Lanel4:
AES722. Lanel5: AES808B.
100
^
Figure 3.13 Autorad after probing with blacjx-M-is/ISEcpl of

blotted PFGE from Fig. 3.12. Lanel: Marker. Lane2: AES8.
Lane3: AES48. Lane4: AES135. Lane5: AES140. Lane6:
LanelO: AES279. Lanel 1: AES280. Lanel2: AES281. Lanel3:
AES506. Lane 14: AES722. Lanel5: AES808B.
3.2.4 Typing of K. pneumoniae by RAPD
Typing of 80 K. pneumoniae isolates by using RAPD technique are illustrated
in Figure 3.14. The K. pneumoniae isolates can be divided into 6 clusters
according to the Pearson correlation test that was performed using GelCompar
software. Members of cluster 2 (n=32) displayed 85% similarity and 34/41
(82.9%) were only collected from patients in Tripoli and Benghazi and
included sites such as blood, urine, sputum, pulmonary, CVL, pus samples,
maternity hospital and bum and plastic surgery centre of Tripoli. Isolates of
this cluster were collected as swabs from the hospital environments and also
included the non-hospital environmental isolates. Cluster one included the
isolates AES 135 and AES 140, AES 172 and AES 178 that appeared clonal
when Xbal digestion was used (see section 3.2.5). Cluster 1 (n=26) also
showed high similarity between members (90%), and 19/26 (73%) of the
isolates were collected from blood, urine, sepsis and embilica samples, they
were also cultured from maternity ward infections and bum ward infections.
Isolates in cluster 2 were found in the hospital environments (bedsides, baby
incubators, vacuum of suction machines, suction machine tubes and floor of
toilets). One member of cluster 2 was isolated from the largest Benghazi Lake
which is considered highly polluted. Members of cluster 4 (n=3) resembles
cluster 3 as all members of this cluster were isolates collected from patients.
Cluster 3 is composed of 6 members collected from a Tripoli maternity
hospital and isolates AES982 and AES985 are clonal. Members of cluster 4
include two isolates (AES225 and AES261) that (by Xbal digestion of the
102
whole DNA) are clonal. Cluster 5 (n=6) includes isolates collected from
patients and in addition to the high similarities (95%) between these members,
they were also all positive for blacrx-u group 1.Cluster 6 (n=7) was different
from the other clusters as members of that cluster share very low similarities
(30%).
103
909
ST 509 Pnumoniae
808B
968
ST 486 Pnumoniae Pnumoniae

809E 225 (PFGE)
192 ST 101
Pnumoniae
-w
0 .6 261 (PFGE)
Cluster 4
Pnumoniae
985 (PFGE)
ST 70 Pnumoniae
K:----- 98? (PFGF)
Pnumoniae
1053 (PFGE)
9ZDI Pnumoniae
ST 511
5Z0I
666 817 (PFGE)
8ZDI
116
6IZ
98
»6 ST 101 Pnumoniae
917
802 ST 15 Pnumoniae
Pnumoniae
1029 (PFGE)
187 (PFGE)
ST 15 Pnumoniae
L9
99 74 (PFGE)
ST 15 Pnumoniae
99
ST 147 Pnumoniae
48 (PFGE)
"wT
811
tl I Pnumoniae
178 (PFGE) Pnumoniae
172 (PFGE)
892
960 (
966
Y1SI
1/2
ST 29 Pnumoniae
140 (PFGE)
90S
£6I pnumoniaea
135 (PFGE) K. pnumoniae
POO i ST 111
1004(1)
196
092
z sa d v a cismva
3.2.5 M olecular typing of K. pneumoniae
PFGE of Xbal digests of 28 K. pneumoniae isolates is shown in Figures 3.15
& 3.16 and the corresponding dendrograms shown in Figures 3.17 & 3.18.
These results show that some isolates of K. pneumoniae are clonally related
(>0.95) despite the different site of collection. Isolates AES135 and AES140
are clonal despite the fact that they were collected from two different
hospitals; K. pneumoniae isolate AES 135 was from a blood sample from a
hospital in Benghazi city whereas K. pneumoniae isolate AES 140 was from a
urine sample from a patient in a hospital from a village near Benghazi. Isolates
AES 172 and AES 178 are clonal. Isolate AES 172 was from a baby incubator
and isolate AES 178 was collected from a vacuum suction machine. These two
clonal isolates were found in the neonatal ICU in Benghazi Paediatric hospital.
The results also show that K. pneumoniae isolates; AES273 and AES260 share
a high-level of similarities (>0.90) and were collected from blood and
umbilical samples, respectively. These two samples were collected from two
different patients; however, the patients were admitted to the same hospital but
not the same ward revealing the potential spread of the same clone within the
hospital. K. pneumoniae isolates AES506 and AES 1013 also share high
similarities (>80%) despite being collected from two different hospitals in
Tripoli; AES506 was collected from a suction machine tube in Tripoli
Paediatric hospital, while AES 1013 was from a patient admitted to Tripoli
burn and plastic surgery centre of Tripoli. The other isolates of K. pneumoniae
105
that were examined by PFGE shared low level of similarities (<75%) showing
that many strains of K. pneumoniae in Libya played a significant role in the
spread of infection and antibiotic resistance genes.
400 kb
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 3.15 PFGE of Xbal digests of K. pneumoniae genomic

DNA. Lanel: Marker. Lane2: AES48. Lane3: AES74. Lane4:
Lane8: AES 187. Lane9: AES225. LanelO: AES261. Lanel 1:
AES 1029. Lanel5: AES 1053.
106
350 kb
300 kb
250 kb
150 kb
100 kb
50 kb
Figure 3.16 PFGE of Xbal digests of K. pneumoniae genomic

AES961.
107
1053 (14) [
1029 (13)
4 8 (1)
985 (12)
9 8 2 (11)
1 40 (4 ) ■ .m i l i it
135 (3) 1 IM K
1 78 (6)
172 (5 )
187 (7) c i m H
8 1 7 (10) IB M
7 4 (2) o h i h
261 (9) H U B
2 2 5 (8)
0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
Figure 3.17 Dendrogram of PFGE gel showing Xbal digested DNA

from K. pneumoniae isolates. Lanel: Marker. Lane2: AES48. Lane3:
AES 178. Lane8: AES187. Lane9: AES225. LanelO: AES261. Lanel 1:
AES817. Lanel2: AES982. Lanel3: AES985. Lanel4: AES1029.
Lanel5: AES 1053.
108
961 (13) H tM s ...
975(7) M m I i '1 S M M M i
1026 (11)
1004 (9) I i i m m
9 77 (8) W) t t M i - m m
1013 (10) K2
506 (6)
273 (4 )
2 60 (3)
2 03 (2)
7 3 (1)
275 (5)
1028 (12)
0.50 0.60 0.70 0.80 0.90 1.00
Figure 3.18 Dendrogram of PFGE gel showing Xbal digested

DNA from K. pneumoniae isolates. Lanel: Marker. Lane2: AES73.
Lane3: AES203. Lane4: AES260. Lane5: AES273. Lane6:
LanelO: AES1004. Lanel 1: AES1013. Lanel2: AES1026. Lanel3:
AES 1028. Lanel4: AES961.
109
3.2.6 Multi-locus sequence typing (MLST)
Representative isolates from RAPD-clusters were subjected to MLST. PCR
experiments yielded PCR products of all housekeeping genes (see Appendix
B). Generally, using RAPD fingerprinting typing method, similar RAPD-types
gives similar sequence types and different RAPD-types give rise to different
sequence types (STs). Sequencing of housekeeping genes of all 12
representative isolates of K. pneumoniae showed the occurrence of 9 sequence
types among all isolates tested. The sequence types found were ST15, ST111,
ST29, ST147, ST511, ST70, ST101, ST486 and ST509. ST15, ST111, ST29,
ST 147, ST70 and ST101 were among the clinical isolates whereas ST511,
ST486 and ST509 were non-hospital environmental isolates from Benghazi. It
is worthy o f note that three isolates had ST15; AES59, AES74 and AES1029.
AES Isolates 59 and 74 were collected from mechanical ventilators from an
ICU ward of the 7th of October hospital in Benghazi, whereas AES 1029 was a
clinical isolate from a patient admitted to a Bum ward in Alkhadra hospital in
Tripoli. These STs were from clusters which shared more than 90%
similarities and part of one large cluster which included 17 members. One
exception was observed, with ST101 being observed in two unrelated RAPD-
clusters sharing less than 60% similarities. The isolates that had ST101 were;
AES261 which was a clinical isolate recovered from a blood sample from Al-
Jamhoryia hospital in Benghazi and AES isolate 917 which was from a curtain
on an ICU ward in Al-Jala hospital in Benghazi. Nevertheless, both isolates
110
were detected positive for blaax-u group 1. The most frequently observed
sequence type was ST15, which has earlier been described in 6 /t f c r x - M - i5 -
producing K. pneumoniae (Damjanova et al., 2008). Also, ST15 is a single
locus variant (SLV) of STM, which has been described in blacjx-u, blaKPc
and &/#ndm-i producing K. pneumoniae (Hrabak et al., 2009; Oteo et al., 2009;
Kitchel et al., 2009; Samuelsen et al., 2011) Two other sequence types,
ST 147 and ST101 have also been linked to the dissemination of blacrx-u in
previous reports. (Hrabak et al., 2009; Damjanova et al., 2008). ST29 was a
clinical isolate from blood and was also positive for blacrx-u-\5 - This ST has
earlier been described in extended-spectrum cephalosporin-resistant isolates,
but has not been frequently reported recently (Diancourt et al., 2005). ST70
was a clinical isolate from Tripoli maternity hospital and positive for blacrx-u
group 1, while ST111 was a clinical isolate recovered from a patient in bum
and plastic surgery centre of Tripoli, and was also positive for blacrxu group
1. ST70 and ST111 have not been associated with dissemination of CTX-M-
producing K. pneumoniae in previous reports, and are not closely related to
any of the main epidemic clones. The novel sequence type
ST511(http://www.pasteur.fr/cgi-bin/genopole/ PF 8 /mlstdbnet.pl ?file=klebs_
profiles.xml&page=profileinfo&st=511) is an environmental isolate cultured
from a swab collected from one of the Benghazi streets. This isolate carries a
plasmid mediated blact x - m - i s and is a double-locus variant of ST35 and ST36
which have both recently been described in CTX-M-producers (Oteo et al.,
2009). Two environmental isolates were new sequence types, ST486
111
(http://www.pasteur.fr/cgibin/genopole/PF 8 /mlstdbnet.pl?flle-klebs_profiles.x
ml&page=profileinfo&st=486) and ST509 (http://www.pasteur.fr/cgi-
bin/genopole/PF 8 /mlstdbnepl?file=klebs_profiles.xml&page=profileinfo&st=
509). These isolates were also cultured from a swab collected from two
different roads in Benghazi.
3.2.7 Detection of chromosomally and plasmid mediated /> /0 c t x - m groupl
Probing of PFGE o f Xbal digests of K. pneumoniae (figures 3.15 & 3.16) with
radio-labelled / j / a c t x -m - i s template DNA is shown in (Figures 3.19 & 3.20). K.

pneumoniae AES48, AES 135 and AES 140 possesses four copies of blacix-u
group 1 in different locations including the various plasmids. K. pneumoniae
AES74, AES 172, AES178, AES225, AES1029, AES1053, AES260, AES273,
AES275 and AES 1026 possess two copies of blact x - m groupl genes. Only one
copy of blac t x - m group 1 gene was detected in K. pneumoniae AES261,
AES817, AES982, AES985, AES73, AES506 & AES1004. The incidence of
more than one copy of blacix-u group 1 gene in some isolates of K.
pneumoniae might raise the question of how can blacix-u group 1 genes move
within the genome of K. pneumoniae, Such movement could be facilitated by
the active presence of ISEcpl which can mobilise blacix-u group 1. Digestion
with Xba/ does not discriminate plasmid from chromosome and therefore the
bands seen in Figures 3.19 & 3.20 can only refer to the number of copies of
blacix-u group 1 and not their genetic location.
112
3.2.8 Transconjugation Experiments
Transconjugation experiments on 51 K. pneumoniae positive for blacix-u
groupl showed that successful transfer of resistance occurred in 27/51
(52.9%). PCR analysis on transconjugants confirmed the movement of blacix-
m group 1 and it is promoter sequence ISEcpl from parents of K. pneumoniae
to transconjugants (E. coli) (Figures 3.21 & 3.22). Sequencing of these alleles
showed the occurrence of blac\x-u groupl and ISEcpl in the new generation
of transconjugants and further confirmed the movement capability of blac t x - m
groupl/IS£cp/ from parents to recipients, indicating the role of conjugative
plasmids in transfer. Some transconjugation experiments failed to transfer
blac t x - m groupl assuming the non-conjugative plasmid location of blac t x - m
groupl and/or ISEcpl or the occurrence of one copy of chromosomal located
blacjx-Mgroup 1 .
113
Figure 3.19 Autorad after probing with blacix-u-\s of blotted
PFGE from Fig. 3.15. Lanel: Marker. Lane2: AES48. Lane3:
AES74. Lane4: AES135. Lane5: AES140. Lane6 : AES172.
Lane7: AES 178. Lane8 : AES2187. Lane9: AES225. LanelO:
AES261. Lanel 1: AES817. Lanel2: AES982. Lanel3: AES985.
Lane 14: AES1029. Lanel5: AES1053.
Figure 3.20 Autorad after probing with bla
c t x - m - 15 o f blotted
PFGE from Fig. 3.16. Lanel: Marker. Lane2: AES73. Lane3:
AES203. Lane4: AES260. Lane5: AES273. Lane6 : AES275.
Lane7: AES506. Lane8 : AES975. Lane9: AES977. LanelO:
AES 1004. Lanel 1: AES1013. Lanel2: AES1026. Lanel3:
AES1028. Lane 14: AES961.
115
Figure 3.21 Detection ofblacix-u groupl/IS£c/?7 in GFP E. coli
transconjugants of K. pneumoniae AES isolates. Lanel: Marker. Lane2:
AES74T. Lane3: AES178T. Lane4: AES261T. Lane5: AES268T. Lane6 :
AES273T. Lane7: AES274T. Lane8 : AES280T. Lane9: AES970T. LanelO:
AES975T. Lanel 1: AES984T. Lanel2: AES1001T. Lanel3: negative
control.
Figure 3.22 Detection of the occurrence of an intact (2-5) and

disrupted (7-10) copies of IS7sc/?7 in GFP E. coli transconjugants of
K. pneumoniae AES isolates. Lanel: Marker. Lane2: AES74T.
Lane3: AES172T. Lane4: AES178T. Lane5: AES268. Lane6 :
Marker. Lane7: AES74T. Lane8 : AES172T. Lane9: AES178T.
LanelO: AES268T.
3.2.9 Detection of plasmid mediated blaCjx-M groupl in parents and
transconjugants
PFGE separation of S1 endonuclease digestion of genomic DNA from a subset
of 6 parents o f K. pneumoniae and their 6 recipients of E. coli are shown in
Figure 3.23. The result o f the probed PFGE gel with a custom made blacix-M-
15 probe is shown in Figure 3.24. Probing of the PFGE gel showed that blacix-
m groupl have successfully transferred to E. coli as the recipient. The results
clearly confirm the plasmid location and also demonstrated that the plasmid
carrying blacix-u groupl has moved, more or less, unaltered. Intriguingly, the
data from Fig. 3.24 also shows that some of the copies of blacix-u groupl are
chromosomal a phenomenon not well cited in the literature.
3.2.10 Detection of the movement of ISEcpl from parents to

transconjugants
S1 endonuclease digestion and separation of genomic DNA by PFGE of a
selection of parents and transconjugants are illustrated in Figure 3.25. The
results of probing of the PFGE gel with radio-labelled blacix-u-\ ÎÊcpl

5
genes are shown in Figure 3.26. These results show the same size plasmids in
parents and transconjugants. The results clearly demonstrate the capability of
clinical and non-clinical isolates of Libyan K. pneumoniae to acquire & /« c t x -m
groupl/I and to confer such a resistance mechanism to recipients of E.
coli. blac t x - m groupl /I SEcpl have been detected on a plasmid of 300kb in
isolates AES74, AES 135, AES 140 and AES 141 and their respective
recipients, blacix-u group l/IS£c/?/ was also detected on a lOOkb plasmid in
117
AES 172, AES172T, AES 178 and AES178T. AES48 demonstrates the
incidence of 5 copies of blacix-ugroup 1/lSEcpl on plasmids of different sizes
-50, 100, 200, 250 and 300kb.
400 kb
350 kb |
300 kb 1 m
250 kb
200 kb
150 kb
100 kb
50 kb
m B *' C T y
Figure 3.23 PFGE of S1 digests of K. pneumoniae and GFP

transconjugants. Lanel: Marker. Lane2: AES74. Lane3:
AES74T. Lane4: AES135. Lane5: AES135T. Lane6 : AES140.
Lane7: AES 1401. Lane8 : AES2141. Lane9: AES141T.
LanelO: AES172. Lanel 1: AES172T. Lanel2: AES178.
Lanel3: AES178T. Lane 14: AES48 (positive control). Lanel5:
5738 (positive control).
118
Figure 3.24 Autorad after probing with blacix-M-is of
blotted PFGE from Fig. 3.23. Lanel: Marker. Lane2:
AES74. Lane3: AES74T. Lane4: AES135. Lane5:
AES135T. Lane6 : AES140. Lane7: AES140T. Lane8 :
AES2141. Lane9: AES141T. LanelO: AES172. Lanel 1:
AES172T. Lanel2: AES178. Lanel3: AES178T. Lanel4:
AES48 (positive control). Lanel5: 5738 (positive control).
119
400 kb
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 3.25 PFGE of S1 digests of K. pneumoniae and GFP

transconjugants. Lanel: Marker. Lane2: AES48 (positive
control). Lane3: AES 1052 (Negative control). Lane4: AES74.
Lane5: AES74T. Lane6 : AES 135. Lane7: AES135T. Lane8 :
AES2140. Lane9: AES140T. LanelO: AES141. Lanel 1:
AES141T. Lane 12: AES172. Lanel3: AES172T. Lanel4:
AES 178. Lanel5: AES178T.
120
Figure 3.26 Autorad after probing with blacTx-M-\ Êcpl
5 of
blotted gel from Fig. 3.25. Lanel: Marker. Lane2: AES48
(positive control). Lane3: AES 1052 (Negative control). Lane4:
AES74. Lane5: AES74T. Lane6 : AES135. Lane7: AES135T.
Lane8 : AES2140. Lane9: AES HOT. LanelO: AES141. Lanel 1:
AES141T. Lanel 2: AES172. Lanel3: AES172T. Lanel4:
AES 178. Lanel5: AES178T. (T: transconjugate of respective
parent)
121
3.2.11 Plasmid Typing
PCR reactions failed to produce any inc/rep PCR products of the K.

pneumoniae plasmids; nevertheless, inc/rep PCR products were detected on
the positive control reference plasmids. These results suggest that these
plasmids are non-typeable. They also suggest that the plasmids responsible for
carrying 6 /< 2 c t x - m -1 5 and blacix-ugroupl are significantly different from those
already characterised by Carattoli et al., 2005 which to date is considered the

most recent and applicable system for detecting conjugative plasmids.
3.2.12 Detection of mobile genetic elements
3.2.12.1 Class 1 integrons
The results of PCR reactions yielded PCR products of different sizes and
copies in 20/ 22 (90.90 %) randomly selected isolates (Figure 3.27). Isolates
AES 8 , AES85, AES179, AES198, AES271A, AES280, AES135 and AES140
produce a lkb class 1 integrons whereas isolates AES48, AES59, AES 6 6 and
AES74 were positive for a 1.5kb integron. Two copies of class 1 integrons
were found in K. pneumoniae isolate AES48. Sequencing of these alleles
showed 4 different genetic contexts (B . 8 - B.17). The differences between
these integrons depend on the number and type of gene cassettes embedded in
these integrons. K. pneumoniae isolates AES 179, AES 198, AES271, AES280,
AES 8 , AES 135 and AES 140 share the same class 1 integron genetic context.
This integron is composed of an integrase gene and dihydrofolate reductase
genes that confers resistance to trimethoprim (dfrA?>0), and resistance to
122
sulphamethoxazole (qacENsull) (Figure 3.28B). Integron of AES 135 was
submitted to the gene bank and assigned accession numbers; HE613850.1,
HE613852.1, HE613851.1 and HE613853.1. Class 1 integrons detected in K.

pneumoniae isolates AES59, AES 66 and AES74 were found sharing the same
genetic context; an integrase gene and a dihydrofolate reductase type VII
(dfrAM) which confer resistance to trimethoprim and an aminoglycoside-3
adenyltransferase resistance gene (aadAS) flanked with the conserved region
qacEA/sull (Figure 3.28C). The occurrence of 3 destinct integrons was
identified in K. pneumoniae isolates AES48 (Figure 3.28D) and AES85
(Figure 3.28A). AES48 had a class 1 integron composed of an integrase gene,
dfrAM and aadA2 which is known to confer resistance to streptomycin and
spectinomycin, and qacENsul\. Only one gene cassette, dfrAl, was found
embedded in the integron of AES85 (Figure 3.28).
Figure 3.27 Amplification of the classical class 1 integrons. Lanel:

Marker. Lane2: AES8 . Lane3: AES25. Lane4: AES48. Lane5: AES59.
Lane6 : AES64. Lane7: AES6 6 . Lane8 : AES74. Lane9: AES85. LanelO:
AES 170. Lanel 1: AES172. Lanel2: AES178. Lanel3: AES179.
Lane 14: AS198. Lanel5: AES271. Lanel6 : AES275. Lanel7: AES280.
Lanel 8 : Marker
123
A
///////////S S ///S //S *

'//////////*■*/&//*
'//A r /////////////s ////////////////////<
/// / / / / / / / / / / / / / / / / / ;
B
777777777777777777? 77777777777777777777.,
'S S S S S S M S S S fy Z S S S S ;
's s s s s g y fffifs s s s s .
zm m k z 'S /S S /Z /S /S S /S S /S S S S i
t/S /////S /S /////S /S *///////////////////<
c
* 77777777/ 77*77 >777777 7J777777777*777777777?
Y / / / / / / / / / / S / / / / / S S / S /< / S / S / / / / S / / / / S / / S S / s
fn tfi audAS ^"/jfacEA'z SSSSS^}*y£;t'%SSSSs

/* s s s s AcJ Y M L A s s s s z *
' ^ jS H r s s s z s s s s s s s s s s z s s z s s
iim
r ssssssssssssssssss. v
\:\dadA2-;'- :■mkm&B&zz,
1 1
m
Figure 3.28 Genetic context of 6 class 1 integrons found in K.

pneumoniae isolates. A: Class 1 integron from AES85. B: Class 1
integron from isolates; AES 198, AES 179, AES271, AES280, AES8,
AES 135 & AES 140. C: Class 1 integron from isolates; AES74,
AES66 & AES59. D: Class 1 integron from isolate; AES48.
124
3.2.12.2 Identification of Tn402 transposons
Amplification of tniC gene (a marker for Tn402) was detected in 14/20 (70 %)
isolates randomly examined (Figure 3.29). Sequencing of PCR products of 3
isolates of K. pneumoniae showed the occurrence of two different types of
Tn402 type transposons in three isolates of K. pneumoniae - AES 135, AES 197
and AES258. Isolate AES 135 was also positive for the presence of class 1
integron (Figure 3.30). The transposon was found composed of an integrase
gene, the trimethoprim resistance gene (<#E430), qacE, and tniC (Figure 3.30).
These results show the presence of Tn402 transposons in both clinical and
non-clinical isolates of K. pneumoniae (listed in Table 3.3). As judged by
colony blotting only 22/80 (27.5 %) were positive for tniC type transposons,
and 16/22 (72.7 %) of these transposon positive isolates were also positive for
blacxx-u groupl. In spite of the low occurrence of Tn402 compared with class
1 integrons, PCR data indicates that some isolates possess more than one copy
of tniC.
125
Figure 3.29 Detection of Tn402 type transposons among K.
pneumoniae isolates. Lanel: Marker. Lane2: AES 135. Lane3:
AES 140. Lane4: AES141. Lane5: AES157A. Lane6: AES170.
Lane7: AES 198. Lane8: AES258.
A
7777 ..^
s/sssss?fs/J7?//////.
y///////////////////.
Figure 3.30 Genetic contexts of two Tn402 type transposons found in K.
pneumoniae isolates. A: transposon from AES 135
126
3.2.12.3 Transposase Encoding Genes
3.2.12.3.1 PFGE of S I genomic digests and probing with tniC
PFGE of SI digest of genomic DNA and separation of plasmid according to
size are shown in Figures 3.31 & 3.33. Probing of the PFGE gel with radio
labelled tniC gene is shown in Figures 3.32 & 3.34. Tn402 was detected 13
isolates of K. pneumoniae, 5/13 (38.5 %) carry two copies of the transposon
on 6 different sizes of plasmids of approximately 10, 15, 50, 60, 75 and 100
kb. Two isolates, AES 135 and AES 140, carry the transposon on a plasmid of
250kb and another isolate, AES157A, carries the transposon on a plasmid of
175kb. Isolates AES 179, AES 198 and AES258 have a Tn402 transposon
carried on a plasmid of 200kb. These transposons can act as gene capturing
systems and contribute in the dissemination of antibiotic resistance genes by
carrying genes responsible for conferring antibiotic resistance as part of class
1 integrons (Sajjad et al., 2011).
127
Figure 3.31 PFGE of £1 digests of K. pneumoniae. Lanel: Marker. Lane2:
AES7. Lane3: AES8. Lane4: AES10. Lane5: AES25. Lane6: AES27. Lane7:
AES48. Lane8: AES53. Lane9: AES59. LanelO: AES64. Lanel 1: AES66.
Lanel2: AES67. Lanel3: AES68.
100 kb
■
50 kb
Figure 3.32 Autorad after probing with tniC of blotted gel from Fig. 3.31.
Lanel: Marker. Lane2: AES7. Lane3: AES8. Lane4: AES10. Lane5: AES25.
Lane6: AES27. Lane7: AES48. Lane8: AES53. Lane9: AES59. LanelO:
AES64. Lanel 1: AES66. Lanel2: AES67. Lanel3: AES68.
128
400 kb
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb 1 2 3 4 5 6 7 8 9 10 11
Figure 3.33 PFGE of .SI digests of K. pneumoniae genomic

AES 140. Lane5: AES 152. Lane6: AES 157. Lane7: AES 172.
400 kb
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 3.34 Autorad after probing with tniC of blotted PFGE

gel from Fig. 3.33. Lanel: Marker. Lane2. AES7. Lane3:
Lane7: AES172. Lane8: AES2178. Lane9. AES179. LanelO:
AES198. Lanel 1: AES258.
3.2.12.4 Detection of ISCR Elements
Probing of K. pneumoniae isolates with ISC7?2 genes is presented in (Table
3.3) and (Figures 3.35A, 3.35B, 3.36A & 3.36B). 17 isolates were positive for
ISC7?2. Of these, 12/17 (70.5 %) were also positive for &/<2c t x -m groupl. 5/17
(29.41 %) were also positive for Tn402. 13/17 (76.47 %) K. pneumoniae
isolates possessing ISC7?2 were from patients whereas, 3/17 (17.6 %) were
isolates found in the hospital environment. Only one K. pneumoniae strain
collected from the broader Benghazi environment was positive for blacxx-u
groupl, ISEcpl and ISC7?2 and also showed successful transconjugation.
A B
Figure 3.35 Probing of blotted K. pneumoniae isolates (1-47) with the
ISCR2 gene. A: K. pneumoniae isolates on MacConkey Agar. B:
Autorad of blotting after probing with ISC7?2 gene.
130
A B
Figure 3.36 Probing of blotted K. pneumoniae isolates (48-80) with
ISCi?2 gene. A: K. pneumoniae isolates on MacConkey Agar. B:
Autorad of northern blotting after probing blotted plate A with
ISCR2 gene
131
3.3 Discussion
Due to the fact that there is little information on the current rate of infection
and the spread o f resistant strains of Gram-negative bacteria in Libya, this
study was conducted to examine the resistance mechanisms (in some cases, in
detail, a randomly selected subset) o f K. pneumoniae isolates from Tripoli and
Benghazi collected from the clinical settings and the environment outside the
hospitals.
In addition to the fact that this study represents the first molecular analysis of
antibiotic resistance on Gram-negative bacteria from Libya and in particular
K. pneumonia, it has also major findings. The incidence of blacix-u groupl
type ESBLs and the prevalence of chromosomally and plasmid mediated
blaCTx-MA5i\SEcpl and blacix-u groupl among K. pneumoniae isolates are a
key factor for their resistance. In addition these isolates are able to confer and
express third generation cephalosporin resistance to sensitive E. coli via
conjugative plasmids. Furthermore, the occurrence of clonally related isolates,
in addition to the occurrence o f new sequence types among K. pneumoniae is
a major finding of this study. The involvement of class 1 integrons and Tn402
type transposons as genetic mobile elements in some of these isolates aid
spread of antibiotic resistance genes in Libyan hospitals.
The prevalence rate of CTX-M groupl genes in this study is markedly higher
than the percentage reported in Algeria, Europe, USA and Canada (Messai et
132
al., 2008). Figures 3.10, 3.11, 3.13, 3.19, 3.20, 3.24 and 3.26 clearly
demonstrate the incidence of blacxx-u growpl/lSEcpJ in clinical, non-clinical
and environmental isolates o f K. pneumoniae. It shows the dissemination of
MDR K. pneumoniae in the clinical settings more in than the environment
outside the hospitals. These findings are in accordance with the results of
Mamlouk et al., 2006, who reported a high incidence of blacxx-u groupl in
clinical specimens in Tunisia. The increasingly spread of blacxx-u- groupl in
Libya is likely due to the high consumption of the antibiotics cefotaxime and
ceftazidime in the last ten years to treat infections in Libya. It might also be
due to the lack of hygiene in hospital, such as hand hygiene, sterilisation,
infection control and lack of surveillance programmes that is desperately
lacking in Libya.
SHV and TEM type ESBL genes have also been found prevalent as high
percentages among clinical isolates (78.8% and 77.7% respectively), blasm
and blajxLu were also detected in the non-clinical isolates collected from
floors, curtains, hospital equipment, and surfaces of baby incubators. SHV
type ESBLs were also detected in environmental strains collected from streets.
These findings illustrate the increased level of resistance in clinical isolates of
K. pneumoniae and also highlight the depressing reality that this resistance is
widespread across Libya and that resistance, in this instance, has got very little
to do with the consumption of antibiotics.
133
blacjx-M groupl was detected carried on 7 different plasmid sizes in 14
isolates of K. pneumoniae. Ten isolates were clinical samples, 3 were from the
hospital environment and one isolate was from Benghazi streets. Overall, the
occurrence o f plasmid mediated blacxx-u group lIS£cp7 seems to be higher in
the clinical settings, blacix-u group 1/lSEcpl was found in 6 different hospitals
in Tripoli and Benghazi on different plasmid sizes and locations, blacxx-u
group 1/lSEcpl was found on a plasmid of 300kb in 4 clinical samples, two of
which were clonally related from two different hospitals in Benghazi;
Jamhoryia and Kwaifia hospitals. These findings show the incidence of
blacxx-u group \/\SEcpl in clonally and non-clonally related isolates of K.
pneumoniae. This group of ESBLs was also located on a plasmid of 75kb in 3
clinical isolates of K. pneumoniae (AES274, AES280 and AES281) and on
plasmids of lOOkb and 275kb in the clinical isolates AES275 and AES48
respectively that were collected from Jamhoryia hospital. Although the same
gene, with its promoter sequence was found on a plasmid of 150kb in K.
pneumoniae clinical isolates AES982 and AES970 collected from Al-Jala
Maternity hospital in Tripoli, they were found on the same plasmid size in the
hospital environmental isolate AES506 swabbed from Al-Jala Paediatric
hospital in Tripoli. It is worth mentioning that Al-Jala Paediatric hospital is
located in Tripoli city centre and next to Al-Jala Maternity hospital. This
might explain the occurrence of the blacxx-u groupl in clinical isolates and the
hospital environmental isolates despite being clonally unrelated according to
RAPD test.
134
[
A plasmid mediated blacxx-u group 1/lSEcpl was detected on a large plasmid,
sized 425kb in a K. pneumoniae isolated from one of Benghazi streets. A
possible explanation for the relatively low frequency of plasmid or
chromosomally mediated blacix-u groupl in the streets could be because of
the effect of the environment conditions outside the clinical settings. The
results of this work are in agreement somewhat with the findings of (Lavollay,
et al., 2006) in terms of the wide range of the occurrence of blacxx-u-\s on
plasmids of different sizes. These results are also consistent with the findings
of the spread of plasmid mediated ESBLs that have been reported in K.
pneumoniae strains in Europe and USA (Gori et al., 1996) and Tunisia (Elhani
et al., 2010). The work described in this section conflicts somewhat with the
findings of Gonullu et al., 2008 who found that most blacxx-u-xs^SEcpl were
found in most cases located on a plasmid of the same size and type - in this
cane IncN. The results of this section are also dissimilar to the work of
(Messai et al., 2008) who reported the prevalence of CTX-M genes on
plasmids of approximately 77kb and 85kb.
Several important clones, which were recently found associated with spread of
blacix-u and/or carbapenemases were described in this study. Hence, the study
provides further support to the assumption that epidemic international clones
are responsible for a substantial part of dissemination of blacxx-u among K.
pneumoniae. Transconjugation and detection of the movement of plasmid
135
mediated blacix-u groupl has been detected in K. pneumoniae ST15, ST29,
ST101 and the new environmental allele ST511. Plasmid mediated blacix-u
groupl has also been detected in K. pneumoniae ST 147, ST111 and ST70. The
spread of blacix-u -\5 producing K. pneumoniae has moreover been discovered
in ST101 and ST 147 in Tunisia (Elhani et al, 2010) and in this case Libyan
patients might serve as a reservoir of such sequence types of K. pneumoniae as
Libyans travel frequently to Tunisia in particular for medical purposes,
cosmetic surgery and other medical necessities.
Determination of class 1 integrons and transposons by different methods
showed the incidence of 5 genetic context forms of class 1 integrons in 12
isolates of K. pneumoniae. Some isolates shared the same genetic context
while others had a different integron each. Isolate AES8 5 was found in a CVL
sample and was positive for blacix-u group 1/lSEcpl and a globally distributed
class 1 integron. The integron found in this isolate contained Inti, dfrAl and
qacEAIsull. Several authors report the incidence of this integron in a number
of clinical isolates - S. typhi serotype Typhi from Jordan, Nepal, Senegal,
Uganda and South Africa (Al-Sanouri et al., 2008; Tamang et al., 2007; Sow
et al., 2007; Krauland et al., 2009). An identical integron was, in addition
found in clinical isolates of E. coli and K. pneumoniae from Sweden (Brolund
et al., 2010), in UTI clinical isolate of E. coli from Korea (Yu et al., 2004) and
in Shigella flexneri from Spanish patients who had visited Kenya.
136
Collectively, the high prevalence and abundance of blacix-u groupl and the
occurence of 6 / « c t x -m - i 5 on its own and in association with ISEcpl, blasuv,
blajEM, classical class 1 integron alone or embedded in transposon Tn402,
indicate that the epidemiology of K. pneumoniae in Libyan hospitals is
complex and probably reflects the existence of a longstanding infection
control problems in each hospital. The data also indicates that resistance
outside the hospital environment and in the community is also an issue.
137
Chapter Four
Characterisation of antibiotic
resistance in E. coli isolates from
Tripoli & Benghazi, Libya
4.1 Introduction
E. coli is a major cause of infections in humans and plays a significant role in
nosocomial and CAIs particularly UTIs and bacteraemia among all ages of
humans (Oteo et al., 2010a; Rogers et al., 2011; Oteo et al., 2010b). ESBLs
emerged in late 1980s causing healthcare associated infections that were now
resistant to extended-spectrum (3-lactamases and have spread worldwide
(Apisamthanarak et al., 2008; Kiratisin et al., 2008). In particular, plasmids
mediated ESBLs. It is a probably the result of the extensive use of p-lactam
antibiotics (Goyal et al., 2009) and the selective pressure of these antibiotics
which has caused the spread of plasmids from one pathogen isolate to another.
blacxx-u genes encode for CTX-M enzymes, these genes are often plasmid
encoded and known as narrow-host range plasmids. CTX-M type enzymes are
among the most prevalent ESBLs in Europe, North America, Asia, Latin
America and Africa (Gonullu et al., 2008). It has been reported in Tunisia,
Algeria, Lebanon and Egypt (Khalaf et al., 2009). This type of ESBLs can be
moved from bacteria to bacteria by means of transferable plasmids via
conjugation. These enzymes, particularly the early ones that were discovered,
preferably hydrolyse cefotaxime more than ceftazidime (Dhanji et al., 2011).
blacix-u -\5 ESBLs is the most frequently reported hydrolysing enzyme in the
UK, Italy, Turkey, Spain, Australia, Kuwait, Lebanon, Algeria and Tunisia
(Randall et al., 2011; Cerquetti et al., 2010; Gonullu et al., 2008; Diaz et al.,
2010; Ensor et al., 2009; Sidjabat et al., 2010; Abbassi et al., 2008; Mohamed-
139
Al-Agmy et al., 2006). The outbreak of clonally related strains of E. coli has
been reported in association with the incidence of ESBLs (Abbassi et al.,
2008; Woodford et al., 2004). In view of the increasing world wide emergence
of ESBLs and because there is no detailed information on the occurrence of
ESBLs in Libya this study was carried out to study the prevalence of antibiotic
resistance in 39 clinical and non-clinical isolates of E. coli collected in 2009
from Tripoli and Benghazi hospitals. This study was also conducted to asses
the incidence of blaax-u group 1 encoding gene along with the mobile genetic
element ISEcpl that facilitates its movement and expression.
The results of this section describe the incidence of E. coli collected from
clinical settings from Tripoli and Benghazi, it also demonstrates the
prevalence of ESBLs among these isolates, particularly of CTX-M group 1
type. This section provides an evidence of the occurrence of chromosomally
and plasmid mediated CTX-M-15 and CTX-M-3 in association with the
insertion sequence ISE c p l.
140
4.2 Results
4.2.1 Characterisation of E. coli isolates and antimicrobial susceptibility

testing
Thirty nine isolates of E. coli were collected in a 4 week period in 2009 from
patients admitted to different wards and ICUs from 10 hospitals in Tripoli and
Benghazi (Table C.l). Some of the isolates were also from hospital
environments such as mechanical ventilators, floors, walls, bedsides and other
parts of the hospitals (see Appendix C). The MIC 50 and MIC90 values are
shown in table 4.1. Ceftazidime showed higher MIC 50 and MIC90 than that of
cefotaxime, low MIC50 and MIC90 was observed for carbapenems whereas
high range was shown for piperacillin/tazobactam and ampicillin. In general,
high-level of resistance was observed towards 3rd generation cephalosporins.
Twenty four out of 39 (61.5 %) were resistant to cefotaxime, 16/39 (41%)
resistant to cefuroxime and 17/39 (43.5%) were resistant to ceftazidime. Few
of the isolates (7/39) (17.9%) were resistant to ciprofloxacin and 2/39 (5%)
and to piperacillin-tazobactam. Those isolates displaying resistance to 3rd
generation cephalosporins also showed resistance to aztreonam, trimethoprim
sulphamethoxazole - 53.8%, and 35.8% respectively.
4.2.2 Detection of TEM, SHV and CTX-M type ESBL genes
Amplification of blajû and blasuv has shown the occurrence of blcijEM in 7
isolates and blasuv in 8 E. coli isolates tested. Amplification of the major
CTX-M groups (1, 2 ,8 ,9 and 26) showed that 23 out of 39 (58.9%) were
141
positive for CTX-M group 1 and only one E. coli isolate gave PCR product for
the CTX-M group 9 (Figures 4.1&4.2). The other CTX-M groups were
negative. The association of the insertion sequence, ISEcpl, with blacjx-M-is
occurred in all cases where CTX-M group 1 was present (Figures 4.3&4.4).
Moreover, three isolates; AES226, AES228 & AES232 showed the occurrence
of an additional CTX-M group 1 gene. Sequencing these PCR products
showed the association of blact x - m group 1 with ISEcpl in 22/26 (84.62%).
The sequencing results of the three different PCR products obtained at 620bp
(isolates; AES226, AES228 & AES232) were positive for CTX-M-3 in
association with ISEcpl in addition to the CTX-M-15/lSEcpl also carried by
these strains. Sequencing results of the single PCR product from the CTX-M
group 9 showed the occurrence of CTX-M-19. Interestingly, a deletion event
has been detected in the insertion sequence located adjacent to /> /< 3 c t x -m - i 5 -
This deletion event has been found in some insertion sequences, it shows that
the ISEcpl is occasionally not intact and probably played a role in the
movement of blacix-u group 1 with some E. coli isolates (Figure 4.5 & 4.6).
4.2.3 Transconjugation experiments
A subset (n=20) of the CTX-M positive E. coli were used to study the
plasmids carrying the CTX-M-15 genes. The results of the transconjugation
experiments using the GFP E. coli as a recipient showed that transconjugation
was observed in 19 out of 20 (95%). Antibiotic resistance profile of E. coli
transconjugants, AES224T, AES226T, AES228T and AES231 showed the
142
occurrence of virtually the same resistance profile from parents to
transconjugants (Table 4.2). Ceftazidime resistant transformants were
confirmed by PCR. Transconjugation was also conducted on the E. coli isolate
positive for CTX-M-19. The plasmid carrying blacix-u -\9 was able to move to
the recipient E. coli conferring ceftazidime which was further confirmed by
PCR.
4.2.3.1 Antibiotic Resistance profile of E. coli CTX-M transconjugants
Antibiotic resistance profile of E. coli transconjugants; AES224T, AES226T,
AES228T, and AES231T are virtually the same as their donor strains. The
original GFP E. coli strain is fully sensitive a part of rifampicin; subsequently
mating E. coli with GFP E. coli (recipient) indicates the movement of
antibiotic resistance mechanism from parents to transconjugants via
conjugative plasmids. The resultant GFP E. coli were resistant to
aminoglycosides, aztreonam, ampicilin, amoxicillin/clavulanate, p-lactam
antibiotics such as cephalosporins, and third generation cephalosporins, they
were sensitive to carbapenems and monobactams. (Table 4.2).
143
Table 4.1. MIC 50 and MIC90 of E. coli isolates
Antibiotic MIC50 MIC90 Range
Ceftazidime 16 32 4 -3 2
Cefotaxime 8 64 2 -6 4
Imipenem 0.5 1 0 .125-1
M eropenem 0.5 1 0.125-1
Aztreonam 16 16 8 -1 6
Piperacillin/T azobactam 8 128 4 -1 2 8
Ciprofloxacin 2 4 0 .5 -8
Ampicillin 16 64 4 -6 4
Gentamicin 16 16 2 -1 6
12 13 14 15 16
Figure 4.1 Multiplex PCR to detect CTX-M groups; 1, 2, 8, 9 & 26 in

E. coli. Lanel: Marker. Lane2: E. coli AES11. Lane3: E. coliAES35.
Lnae4: E. coli AES58. Lane5: E. coli AES120. Lnae6: E. coli
AES128. Lane7: E. coli AES195. Lane8: E. coli AES202. Lane9: E.
coli AES212. Lane 10: E. coli AES224. L nell: E. coli AES226.
Lanel2: E. coli AES227. Lanel3: E. coli AES228. Lnael4: E. coli
AES230. Lnael5: E. coli AES231. Lanel6: E. coli AES232.
144
400 bp
200 bp
■m m m m m m *** - ' ****** mmm.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
F ig u re 4.2 Multiplex PCR to detect CTX-M groups; 1, 2, 8, 9 & 26 in E.

coli. Lanel: Marker. Lane2: E. co li AES237. Lane3: E. co li AES239.
Lane4: E. co li AES240. Lane5: E. coli AES243. Lane6: E. co li AES244.
Lnae7: E. co li AES245. Lnae8: E. coli AES246. Lane9: E. co li AES247.
LanelO: E. c o li AES248. Lanel 1: E. co li AES262. Lanel2: E. co li
AES101. Lane 13: E. c o li AES922. Lanel4: E. coli AES932. Lanel5: E.
co li AES937. Lanel6: E. co li AES938. Lanel7: E. co li AES941. Lanel8:

E. co li AES944. Lnael9: E. c o li AES962. Lane20: E. coli AES964
400 bp
200 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2 0
F ig u re 4.3 Detection of blaCix-u group 1 and ISE cpl in E. coli. Lanel:

Marker. Lane2: E. co li AES11. Lane3: E. co//AES35. Lnae4: E. co li
AES120. Lane5: E. co li AES195. Lnae6: E. co li AES202. Lane7: E. coli
AES224. Lane8: E. co li AES226. Lane9: E. coli AES227. LanelO: E.
co li AES228. Lnell: E. c o li AES230. Lanel2: E. co li AES231. Lanel3:

E. co li AES232. Lnael4: E. co li AES237. Lnael5: E. co li AES239.
Lanel6: E. co li AES240. Lanel7: E. co li AES243. Lanel8: E. coli
AES244. Lane 19: E. coli AES245. Lane20: E. co li AES246.
145
400 bp
200 bp
5 6 7 8
Figure 4.4 Detection ofblacrx-u group 1 and ISEcpl in

E. coli. Lanel: Marker. Lane2: E. coli AES247. Lane3:
E. co//'AES248. Lnae4: E. coli AES262. Lane5: E. coli
AES101. Lnae6: E. coli AES937. Lane7: E. coli
AES941. Lane8: E. coli AES 1006.
1000 bp
800 bp
600 bp
400 bp
200 bp
Figure 4.5 Detection of blacix-u group 1 in association with an

intact copy ofISEcpl in E. coli. Lanel: Marker. Lane2: E. coli
AES11. Lane3: E. co/zAES35. Lnae4: E. coli AES 120. Lane5:
E. coli AES 195. Lnae6: E. coli AES202. Lane7: E. coli
AES224. Lane8: E. coli AES226. Lane9: E. coli AES227.
LanelO: E. coli AES228. Lnel 1: E. coli AES230.
146
1000 bp
800 bp
600 bp
400 bp
200 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Figure 4.6 Detection of blacix-u group 1 in association with ISEcpl in E.

coli. Lanel: Marker. Lane2: E. coli AES231. Lane3: E. coli AES232.
Lane4: E. coli AES237. Lane5: E. coli AES239. Lane6: E. coli AES240.
Lnae7: E. coli AES243. Lane8: E. coli AES244. Lnae9: E. coli AES245.
LanelO: E. coli AES246. Lanel 1: E. coli AES247. Lanel2: E. coli
AES248. Lanel3: E. coli AES262. Lanel4: E. coli AES101A. Lanel5: E.
coli AES937. Lanel6: E. coli AES941
The results of the amplification of blacix-u group 1 and ISEcpl to detect the
occurrence of the full sequence of the insertion sequence ISEcpl showed that
in 12 out of 22 (54.5%) of isolates a deletion event is occurred in the insertion
sequence, The results also demonstrated that 10 out 22 (45.4%) had the full
sequence of ISEcpl. Amplification of blacix-M group 1 and ISEcpl genes in
the transconjugants GFP showed that 18 out of 20 (90%) showed the
occurrence of both genes.
147
Table 4.2 Sensitivity profile of E. coli parents and transconjugants
Antibiotic AES224 AJES224T AES226 AES226T AES228 AES228T AES231 AES231T
Amikacin S 16 S 16 S 16 S 16
Ampicillin >8 >8 >8 >8 >8 >8 >8 >8
Aztreonam >16 >16 >16 >16 >16 >16 >16 >16
Cefotaxime >4 >4 >4 >4 >4 >4 >4 >4
Ceftazidime >16 >8 16 >8 16 >8 16 >8
Cefuroxime >16 >8 >16 >8 >16 >8 >16 >8
Ciprofloxacin >2 S S S S S S S
Gentamicin S >4 >8 >4 >8 >4 >8 >4
Imipenem S S S S S S S S
Meropenem S S S S S S S S
Nitrofurantoin S S S S S S S S
Piperacillin/
S S S s S S S S
Tazobactam
Trimethoprim - S - s - S - S
Trimethopri/
S s s s S S S S
Sulphamethoxazole
Am oxicillin/
16 16 16 16 16 16 16 16
clavulanate
T: Transconjugants
4.2.4 Plasmid typing of ESBL positive E. coli isolates
Typing of a subset of blact x - m group 1 positive E. coli isolates by PCR to
identify the plasmids responsible for the carriage and movement of CTX-M
group 1 and ISEcpl showed that more than one type of plasmids has been
detected in some these isolates. AES224 was positive for incFIA, AES226,
and its transconjugant were found positive for IncFII. AES237 and its
148
transconjugant AES237T were carrying blacjx-u group 1 on Incl plasmid.
AES243 was detected positive for IncF plasmid.
Detection of plasmid mediated A / a c t x - m group 1 and ISEcpl genes in

4 .2 .5
parents and transconjugants of E. coli
PFGE of SI digests of a subset of the whole genomic DNA of parents and
transconjugants of E. coli are shown in figures (4.7&4.9). Probing of PFGE
gels of figures 4.7&4.9 with 6 /« c t x -m - i 5 is illustrated in figures (4.8&4.10).
These results demonstrated the incidence of one copy of blacix-u group 1 in
parents of E. coli isolates; the results of probing provide an evidence of the
movement of 6 / « c t x - m -1 5 and blacix-u group 1 from parents to transconjugants.
During conjugation, on occasions the plasmid carrying blac\x-u -\5 changed in
size, blacix-u -15 has been detected on a plasmid with a size of 100 kb in three
of the parents; AES226, AES228 & AES232 and on 100 kb in 6 /a c t x -m
groupl, AES35, AES227, and AES231; however, during conjugation blacix-u
group 1 was detected on two plasmids (100 and 350 kb) in 3 of the
transconjugants ( AES226T, AES228T & AES232T) and on 100 and 350 kb
of the transconjugants AES227T and AES231T and on a plasmid of 300kb in
transconjugant AES35T. These data show that 6 /a c t x -m - i 5 and bla^x-u groupl
genes have moved either from one plasmid to another larger plasmid during
conjugation or that during the conjugation process the plasmid has acquired
chromosomal DNA or two plasmids (one containing blacix-u groupl gene)
have become co-integrative. blacix-u groupl was also located on a 125kb
149
plasmid in the donor AES237 as well as its corresponding transconjugant, in
one donor (AES224) and its transconjugants blacix-u groupl is present on a
175kb plasmid.
350 kb
300 kb «
250 kb S
200 kb
150 kb
100 kb
50 kb
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure 4.7 PFGE of SI digestion of E. coli parents and

transconjugants. Lanel: Marker, Lane2: E. coli isolate
AES35. Lane3: E. coli AES35T. Lane4: E. coli AES224.
Lane5: E. coli AES224T. Lane6: E. coli AES226. Lane7:
E. coli AES226T. Lane8: E. coli AES227. Lane9: E. coli
AES227T. LanelO: E. coli AES228. Lanel1: E. coli
AES228T. Lane12: E. coli AES231. Lanel3: E. coli
AES231T. Lanel4: E. coli AES237. Lanel5: E. coli
AES237T.
150
350
300
250
200
150
100
50
Figure 4.8 Autorad of E.coli parents and transconjugants

after probing of PFGE gel from fig.4.7 with blac - - -
t x m is
Lanel: Marker, Lane2: E. coli AES35. Lane3: E. coli

AES35T. Lane4: E. coli AES224. Lane5: E. coli AES224T.
Lane6: E. coli AES226. Lane7: E. coli AES226T. Lane8: E.
coli AES227. Lane9: E. coli AES227T. LanelO: E. coli
AES228. Lanel 1: E. coli AES228T. Lanel2: E. coli
AES231. Lanel3: E. coli AES231T. Lane 14: E. coli
AES237. Lanel5: E. coli AES237T.
151
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure 4.9 PFGE of S\ digestion of E. coli parents and

transconjugants. Lanel: Marker, Lane2: E. coli isolate
AES35. Lane3: E. coli AES35T. Lane4: E. coli AES224.
Lane5: E. coli AES224T. Lane6: E. coli AES226. Lane7:
E. coli AES226T. Lane8: E. coli AES227. Lane9: E. coli
AES227T. LanelO: E.coli AES228. Lanel 1: E. coli
AES228T. Lane 12: E.coli AES231. Lanel3: E. coli
AES231T. Lane 14: E.coli AES232. Lanel5: E. coli
AES232T.
152
...v .
■i l l !
350 kb
' ' : ■■- • - • . •■
MMMi
t t
300 kb
m
250 kb
200 kb ■
* mf ' WmM
150 kb
....... —. KHBS
100 kb
50 kb m
ji t )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure 4.10 Autorad of E.coli parents and transconjugants after

probing of the PFGE gel from fig. 4.9 with blacjx-M-isHSEcpl. Lanel:
Marker, Lane2: E. coli AES35. Lane3: E. coli AES35T. Lane4: E. coli
AES224. Lane5: E. coli AES224T. Lane6: E. coli AES226. Lane7: E.
coli AES226T. Lane8: E. coli AES227. Lane9: E. coli AES227T.
LanelO: E. coli AES228. Lanel 1: E. coli AES228T. Lanel2: E. coli
AES231. Lanel3: E. coli AES231T. Lane 14: E. coli AES232. Lanel5:
E. coli AES232T.
153
4.2.6 Typing of E . c o li isolates
PFGE of X ba\ digests of E. coli isolates; AES35, AES224, AES228, AES231,
AES232, AES237, AES240, AES243, AES245, AES246, AES247, AES226,
AES227, AES11, AES202, AES230, AES239, AES244, AES248 & AES262
are shown in (Figures 4.11 and 4.13). The dendrogram of the PFGE pictures
analysis are illustrated in (Figures 4.12 and 4.14). These results showed the
incidence o f 3 groups o f clones among the 20 E. coli isolates examined. One
clonal group, isolates AES226, AES227, AES228, AES232 and AES231,
were clinical and hospital environmental isolates from an ICU as part of a
screen from the ICU o f the Paediatric hospital in Benghazi, these isolates were
slightly different with computer analysis. AES226 and AES232 were urine
samples cultured from two patients admitted to Benghazi peadiatric hospital
whereas, AES227, AES228 and AES231 were cultured from non-clinical
swabs collected from the ICU of the same hospital. E. coli isolates; AES243
and AES245 were also clonal and found in urine samples from two different
patients suggesting either a dominat Libyan clone or cross-infection. Another
two isolates, AES237, AES240, AES246 and AES247 were also clonal despite
being dissimilar by dendrogram. Isolates AES237 and AES 246 were from
urine samples, while isolates AES240 and AES247 were collected from the
corridor and floor of the ICU at the same hospital, this clone (AES237/
AES240) shared more than 90% similarity with isolate AES247 that was
cultured from the floor o f the same ICU. Isolate AES35 was unrelated to the
154
other strains isolated from environmental swabs of the same ICU at the Al-
Jamhoryia hospital, Benghazi.
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 4.11 PFGE of Xbal digestion and separation of

genomic DNA according to size. Lane 1: Marker. Lane 2:
E. coli AES35. Lane 3: E. coli AES224. Lane 4: E. coli
AES228. Lane 5: E. coli AES231. Lane 6: E. coli AES232.
Lane 7: E. coli AES237. Lane 8: E. coli AES240. Lane 9:
E. coli AES243. Lane 10: E. coli AES245. Lane 11: E. coli
AES246. Lane 12: E. coli AES247. Lane 13: E. coli
AES226. Lane 12: E. coli AES227
155
227 (13)
228 ( 12)
2 4 6 (9 ) • I 11 151
2 4 3 (8) t l It 1*1
232 (5 )
231 (4 )
22 8 (3)
247 ( 11)
2 4 0 (7) M |* i
1 11
248 ( 10)
0 80 0 65 0.70 0.75 0.80 0.85 0.90 0.95 1.00
Figure 4.12 Dendrogram of PFGE picture of E. coli isolates fig

(4.11). Lane 1: Marker. Lane 2:E. coli AES35. Lane 3: E. coli
Lane 6: E. coli AES232. Lane 7: E. coli AES237. Lane 8: E.
coli AES240. Lane 9: E. coli AES243. Lane 10: E. coli
Lane 13: E. coli AES226. Lane 12: E. coli AES227
156
Figure 4.13 PFGE of Xbal digestion
350
and separation of genomic DNA 300
according to size. Lane 1: Marker. 250
200
Lane 2: E. coli AES11. Lane 3: E. coli
150
iAES202. Lane 4: E. coli AES230.
Lane 5: E. coli AES239. Lane 6: E. 100
coli AES244. Lane 7: E. coli AES248.

Lane 8: E. coli AES262.
Figure 4.14 Dendrogram of PFGE picture of fig. (4.13).

Lane 1: Marker. Lane 2: E. coli AES11. Lane 3: E. coli
iAES202. Lane 4: E. coli AES230. Lane 5: E. coli AES239.
coli AES262.
157
4.2.7 Detection of chromosomally mediated blacrx-M groupl encoding
gene
Probing o f the PFGE gels from Figures 4.11 & 4.13 with the radio-labelled
blacjx-u-\5 DNA probe is demonstrated in Figures 4.15 and 4.16. These results
show that two copies o f the blacrx-M groupl were detected in isolate 11 but
only one copy o f blacrx-M groupl gene was detected in the other 19 isolates.
b la cix -u groupl was found on a 50kb plasmid in 6 isolates (AES35, AES228,
AES231, AES232, AES226 and AES227), whereas blacrx-M groupl was
carried on a lOOkb in isolates; AES237, AES240, AES246, AES247,
AES239 and AES248. Four isolates (AES11, AES224, AES243, AES245 and
AES230) carry blacrx-M groupl on a plasmid of 125kb. The results in Figures
4.11 and 4.13 showed that isolates; AES35, AES224, AES227, AES231 and
AES237 were confirmed to express plasmid mediated CTX-M groupl genes at
different plasmid sizes; 50, 125, 50, 50 and lOOkb, respectively while isolates
AES226, AES228 and AES232 showed plasmid mediated CTX-M groupl
genes at 50 kb. The results o f probing the PFGE gel of X b a l digests provide
another evidence o f the occurrence o f the CTX-M groupl genes on plasmids
detected in (Figures 4.8 and 4.10).
158
Figure 4.15 Autorad of PFGE gel of fig (4.11) after probing
with CTX-M-15. Lane 1: Marker. Lane 2: E. coli AES35. Lane
3: E. coli AES224. Lane 4: E. coli AES228. Lane 5: E. coli
coli AES245. Lane 11: E. coli AES246. Lane 12: E. coli
AES247. Lane 13: E. coli AES226. Lane 14: E. coli AES227
159
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
1 2 3 5 6 7 8
Figure 4.16 Autorad of PFGE gel of fig. (4.13) after

probing with CTX-M-15. Lane 1: Marker. Lane 2: E. coli
AES11. Lane 3: E. coli iAES202. Lane4: E. coli
AES230. Lane 5: E. coli AES239. Lane6: E. coli
AES244. Lane 7: E. coli AES248. Lane8: E. coli
AES262.
160
4.2.8 Detection of class 1 integrons & Tn4 02 type transposons
The results of amplification of class 1 integrons from a subset of 14 isolates of
E. coli isolates selected according to their resistance to aminoglycosides and
trimethoprim, demonstrated that 7 isolates; AES11, AES237, AES240,
AES243, AES245, AES246 and AES247 out of 15 yielded PCR products of
approx. 2kb. Sequencing of the 2 kb PCR products obtained from isolates;
AES11, AES245 and AES247 revealed the presence of a classical class 1
integron. The genetic context o f the three integrons were exactly the same
containing two gene cassettes; dfrAXl and aadA5 flanked with the integrase
gene (Intll) and the quaternary ammonium compound gene (qacAE), (Figure
4.17). The integron-positive strains were collected from different sources.
Isolates AES 11 and AES245 were from urine samples from patients admitted
to Al-Jamhoriya hospital and Paediatric hospital in Benghazi, whereas isolate
AES247 was from an ICU surface in the Benghazi Paediatric hospital. PCR
experiments performed on these isolates to detect the occurrence of Tn402
type transposons did not detect the desired amplicons.
d frA ll U ddA S • ////A / / / / / / / ////////<

* /////////////////////.
Figure 4.17 Genetic context of class 1 integrons found in Libyan E. coli

isolates. Intll: Integrase gene. dfrA17: Trimethoprim resistance gene.
aadA5: Aminoglycoside resistance gene. QacEA/SuII: Quaternary
ammonium compound resistance gene and sulphonamides’ resistance
gene.
161
4.3 Discussion
This section describes the molecular characterisation of antibiotic resistance in
a random collection of E. coli from Libyan hospitals. Data from this collection
indicate that the spread of c t x -m groupl along with ISEcpl is well
established in Libyan health institutions. The results moreover demonstrate the
occurrence of blact x - m - i s , blacix-u -3 a n d & / « c t x -m - i 9 among the clinical isolates
in addition to blasuv and 6 /<z t e m .-
E. coli isolates collected from both Tripoli and Benghazi hospitals in Libya
showed that multi-antibiotic resistant isolates were found in Benghazi
hospitals, particularly in the Benghazi Paediatric Hospital. Isolates collected
from inpatients (urine, blood and pus samples) and hospital environments
(mechanical ventilators, baby incubators, surfaces, and bed sites) showed
marginally higher rate of resistance to antibiotics, more specifically to third
generation cephalosporins. There was no observable difference in the
resistance rates of E. coli isolates cultured from samples collected from
patients and isolates cultured from the hospital environment. MICs of 13
isolates showed marginally higher MIC values toward ceftazidime than
cefotaxime, this would argue that there is more than one ESBL has contributed
to the resistance mechanism of these isolates. Only 3 isolates displayed higher
MICs values against cefotaxime compared with that of ceftazidime, this may
be attributed to the occurrence of CTX-M type ESBLs, these findings support
the report o f (Yu & Cheng, 2004; Abassi et al., 2008).
162
E. coli isolates screened for the occurrence of ESBLs showed the prevalence
of CTX-X-M group 1 and CTX-M group 9 among these isolates. Detailed
investigation on this group of CTX-M showed the incidence of ^/actx-m
groupl as the most prevalent ESBL in these isolates. Three isolates
demonstrated the occurrence o f blaax-M -\5 and blacix-u-3 , whereas AES 1006
demonstrated the presence of blacix-u -\9 type ESBLs. blacxx-u-i, has been
detected in three isolates; AES226, AES228 and AES232 in addition to
^ C T X -M -1 5 -
The incidence of blacix-u groupl was high (58.9%), this percentage is
considered high and may reflect the longstanding antibiotic pressure on
cephalosporins in particular 3rd generation cephalosporins. A possible
explanation of the widely scattered blacix-u groupl may be attributed to the
horizontal gene transfer and/or due to the role of the insertion sequence
ISEcpl (Abbassi et al., 2008). This is perhaps not too surprising as several
reports showed the global distribution of ^/actx-m-is in Europe, Asia and
Africa (Woodford et al., 2004; Gonullu et al., 2008; Lavollay et al., 2006, Yu
& K. Cheng, 2004; Ramdani-Bouguessa, et al., 2006; Abbassi et al., 2008).
The findings o f this work are in accordance with the records on the
dissemination of CTX-M-15 and CTX-M-3 in E. coli reported by (Ramdani-
Bouguessa, et al., 2006) in Algeria suggesting the enhancement of blacix-u -\5
movement by ISEcpl.
163
ISEcpl gene was determined for blacjx-M-is positive E. coli’, however, a
deletion event has been observed by PCR in 12 out of 22 positive isolates to
IS Ecpl. According to the findings of this work, this deletion event does not
seem to affect the movement o f CTX-M groupl gene from donor cells to
recipients, moreover the IS E cpl either intact or with a deletion event moved
with the p-lactamase gene by transconjugation experiments. It is likely to
responsible for the movement o f blacrx-M groupl within the same strain but to
different plasmid size as shown in E. coli isolates AES226, AES227, AES228,
AES230, AES231 and AES232 (Figures 4.8 and 4.10) The mobility and
expression of CTX-M type ESBLs by IS Ecpl has been proposed by Poirel et
al ., 2003; Abbassi et al ., 2008.
blacrx-M groupl and ISEcpl have been detected in donors and transconjugants
of E. coli on five different plasmid sizes; 100, 175, 300 and 350kb. E. coli
donors showed the occurrence o f blacrx-M groupl and ISEcpl on one plasmid
for each isolate. In E. coli isolates; AES35, AES226, AES227, AES228,
AES231 and AES232, blacrx-M groupl and ISEcpl were detected on two
different plasmid sizes in recipients whereas they were found in one plasmid
location in donors. Interestingly, the data from this study shows the fluidity of
blacrx-M groupl and ISEcpl by mobilising to another plasmid during
conjugation. Such events are rarely reported.
16 4
Three plasmid types have been detected by PCR in E. coli isolates, Incl in
AES237, IncFII in AES226 and IncFIA in AES224. Several reports have
shown that IncF plasmids (IncFII and IncFIA) are responsible for carrying and
facilitating the movement of blacix-u group 1 and ISEcpl element. (Gonullu et
al., 2008; Villa et al., 2010; Lavollay et al., 2006; Partridge et al., 2011).
Amongst all tested isolates for class 1 integrons, one integron composed of
two gene cassettes; dfrAXl and aadA5 and has been previously described
reported from patient suffered from UTI in Australia. The same integron was
also reported in Spain and China among E. coli isolates (Vinue et al., 2008;
Tang et al., 2011).
Typing of E. coli isolates was performed on the basis of the incidence of
ESBLs more specifically 6/<2ctx-m group 1 among these isolates, this typing
resulted in the occurrence of 3 clonal groups clone 1 ( AES226, AES227,
AES228, AES231 and AES232), clone 2 (AES243 and AES245 and clone 3
(AES237, AES240, AES246 and AES247). Members of clone 1 were
collected from patients and the hospital environment, members of clone 2
were from urine samples from two different patients admitted to the same
hospital whereas members of clone 3 from two different locations; isolate
AES237 and AES246 were from a urine samples while isolate AES240 and
AES247 were from the hospital environment of the same hospital. These
findings would suggest that the inter-dissemination of clonal isolates of E. coli
165
in the same hospital is due to longstanding problem and propose earlier
establishment o f the gene pool in this hospital. Clonal dissemination of blacjx-
m group 1 in E. coli has grasped the attention of many investigators to
understand the epidemiology of antibiotic resistance in the clinical settings and
even outside the hospitals to study the contribution of clonal isolates in the
community (Lavollay et al., 2006; Mashana et al., 2011). The findings of this
section are in accordance to somewhat with clonally spread of E. coli strains
harbouring plasmid mediated 6 /< 2 ctx -m -i5 genes reported by (Coque et al.,
2008).
166
Chapter Five
D etection o f blayiM - 2 in
P. aeruginosa from Benghazi

5.1 Introduction
P. aeruginosa is capable of causing internal and external infections to humans
and largely linked with CAIs and HAIs. It contributes by 10 % among all
other bacterial infections in hospitals and is considered as the leading cause of
cross infections; VAP and wound infections (Enoch et al., 2007). P.
aeruginosa antimicrobial resistance is continuing to rise and this is likely
elucidated by the ability of this micro-organism to live in diverse
environments and share genetic information with numerous species of bacteria
that results in withstanding the effect of antimicrobials by means of antibiotic
hydrolysing enzymes in particular MBLs (Walsh et al., 2005), (Gales, et al.,
2003). The acquisition of MBLs by P. aeruginosa is of particular concern due
to the fact that this enzyme confers resistance to all (3-lactams with the sole
exception o f aztreonam. Furthermore, MBL-producing Gram-negative bacteria
are resistant to nearly all antibiotics and have become pan-resistant resulting in
the wide spread o f treatment failure (Poumaras et al., 2003; Yu et al., 2006).
Section 5 deals with the spread of multi-drug resistant isolates of P.
aeruginosa collected from hospitalised patients, hospital environment swabs
in Tripoli and Benghazi. This work focuses on the spread of mobile genetic
elements; class 1 integrons and transposons associated with MBLs in 14 P.
aeruginosa isolates from Libya. The results show the incidence of multi-drug
resistant P. aeruginosa from clinical and no-clinical sources.
168
blay\u-i has been detected in two isolates of P. aeruginosa collected from two
patients admitted to Al-Jalla hospital in Benghazi. Transconjugation
experiments using E. coli J53 and P. aeruginosa PA01 failed to produce any
ceftazidime resistant transformants, blaym-i has been shown to be
chromosomally located in two isolates of P. aeruginosa. The investigation
did not show the presence o f Tn402 that is usually associated with class 1
integrons to facilitate their mobility. The results also showed the incidence of
3 types o f class 1 integrons among 7 isolates of P. aeruginosa. Novel integron
were submitted to the gene bank and assigned the accession numbers;
HE583392.2 and HE583391.2.
169
5.2 Results
P. aeruginosa collected from clinical and non-clinical samples are illustrated
in Table 5.1.
5.2.1 Antibiotic susceptibility testing
Antimicrobial sensitivity testing o f 14 clinical and non-clinical isolates of P.
aeruginosa is shown in Table 5.2. These results show high-level resistance to
gentamicin, imipenem, aztreonam, cefotaxime, ceftazidime, ciprofloxacin,
piperacillin/tazobactam, trimethoprim/sulphamethoxazole, amoxicillin and
ampicillin. P. aeruginosa isolates AES30, AES81 AES83 and AES93 had
MICs above 8 mg/1. Susceptibility of 10 isolates against amikacin and
meropenem was recorded for the other isolates.
5.2.2 Detection of MBLs using Etest
All isolates were subjected to Etest (see section 2.7) using imipenem and
imipenem plus inhibitor (IP/IPI) to identify the presence of any MBLs. The
results showed that P. aeruginosa isolates AES81 and AES83 had high levels
of resistance to imipenem yet was sensitive to the presence of EDTA (IPI) and
thus indicating the presence MICs higher than 16 mg/1 proposing the
production o f a MBL (Figure 5.1).
5.2.3 Detection of MBL encoding genes
PCR experiments were conducted using primers specific for previously
reported MBLs. Data showed the occurrence of 700 bp amplicons from P.
170
aeruginosa isolates AES81 and AES83 (Figure 5.2). The two amplicons
resulted from primers designed to amplify 6/aviM genes. Both sequences
displayed 100% homology to bla\j\u-i that is disseminated worldwide (Walsh,
et al., 2003).
Table 5.1 List of P. aeruginosa used in experiments
P. aeruginosa Site of collection Place of collection
AES30 Urine Al Jamhoryia hospital Benghazi
AES81 Stainless steel container (Chest ward) Al-Jala hospital Benghazi
AES83 Tip o f catheter (ICU) Al-Jala hospital Benghazi
AES89 Floor of toilet (ICU) Al-Jala hospital Benghazi
AES91 Suction machine tube (ICU) Al-Jala hospital Benghazi
AES93 Suction machine outlet Al-Jala hospital Benghazi
AES 146 Floor of toilet (ICU) Al-Jala hospital Benghazi
AES 182 Pus sample Al-Jala hospital Benghazi
AES273A Blood sample Al Jamhoryia hospital Benghazi
AES284 Blood sample Al Jamhoryia hospital Benghazi
AES287 Urine sample Al Jamhoryia hospital Benghazi
AES934 Wound infection Bum and plastic surgery centre Tripoli
171
Table 5.2 : Antibiotic sensitivity testing of clinical and non-clinical
P. aeruginosa_________________________________________ _____
Antibiotic Sensitive Resistant
Amikacin 10/15 6/15
Gentamycin 5/15 11/15
Imipenem 7/15 9/15
M eropenem 10/15 6/15
Cefotaxim e 2/15 14/15
C eftazidim e 6/15 10/15
Aztreonam 2/15 14/15
Trim ethoprim e sulpham ethoxazole 1/15 15/15
Piperacillin tazobactam 7/15 9/15
Ciprofloxacin 6/15 10/15
A m oxicillin 0/15 15/15
Ampicillin 1/15 15/15
Figure 5.1 Etest of P. aeruginosa isolates. A. P. aeruginosaAES81. B. P.

aeruginosaAES83
172
1000 bp
600 bp
600 bp
400 bp
200 bp
Figure 5.2 Detection of Tn402 type transposon, Tn27 and blay\u-i in P.

aeruginosa isolates AES81 and AES83. Lanel: Marker. Lane2: Tn402 in
isolate AES81. Lane3: negative control. Lane4: Tn402 in isolate AES83.
Lane5: negative control. Lane6: Tn21 in isolate AES81. Lane7: negative
control. Lane8: blawu-i in isolate AES81. Lane9: negative control. LanelO:
Tn27 in isolate AES83. Lanel 1: negative control. Lanel2: blaym -2 in isolate
AES83. Lanel3: negative control.
5.2.4 Transconjugation experiments
Transconjugation experiments using J53 and PA01 as recipients were
performed to detect the possible occurrence of blayim-2 on a transferable
plasmid. The mating experiments did not produce any ceftazidime resistant E.
coli or P. aeruginosa transconjugants suggesting that blaym-i in these clinical
isolates of P. aeruginosa is chromosomally located.
173
5.2.5 Typing of P. aeruginosa
PFGE of Spe- 1 digestion of isolates of P. aeruginosa is shown in Figure 5.3.
Dendrogram of Figure 5.3&5.7 ; in particular isolates AES30, AES81, AES83,
AES89, AES91, AES146, AES182, AES273, AES284 and AES287 are
illustrated in Figure 5.4. Typing of P. aeruginosa showed that isolates;
AES89, AES91, AES93, AES 146 and AES 182 are clonal despite being
collected from two geographically distant places. AES81 was collected from
stainless steel container in Chest ward in Al-Jalla hospital in Benghazi
whereas AES83 was from a clinical sample from tip of catheter from a patient
admitted to the ICU of the same hospital. Isolate AES 182 is from a pus sample
and isolate 146 is from the floor of an ICU toilet. P. aeruginosa isolates
AES81 and AES83 are clonal despite the fact that they were from clinical and
non-clinical samples.
5.2.6 Detection of chromosomally and plasmid mediated blaVIM-2
5.2.6.1 Characterization of chromosomally and plasmid mediated blaym - 2
genes
PFEG of S 1 genomic DNA digestion of Pseudomonas aeruginosa isolates is
illustrated in Figure 5.5. Probed PFGE gel of Figures 5.5&5.7 with a custom
made probe of blaym -2 is shown in Figure 5.6&5.8. These results show that
blaym-i is chromosomally mediated in both isolates positive for the MBL
encoding gene, the results o f probing of the PFGE gel of Spe 1 digestion
174
demonstrate that three copies of ^/«vim-2 were detected carried by both isolates
of P. aeruginosa revealing the occurrence of more than one copy on class 1
integrons in each isolate, The bla\j\u-i positive isolates were negative for
Tn402 type transposons that might facilitate the mobility of class 1 integrons
within the chromosome, intracellular or intercellular but the mechanism of
movement of blay\u -2 cannot be attributed to insertion sequences.
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 5.3 PFGE of Spe-1 digestion of 14 isolates of P.

aeruginosa. Lanel: Marker, Lane2: AES81, Lane3: AES83,
Lane4: AES89, Lane5: AES91, Lane6: AES93, Lane7:
AES 146, Lane8: AES 182, Lane9: AES273A, Lane 10:
AES284, Lanel 1: AES287, Lanel2: AES934, Lanel3:
AES988, Lane 14: AES998, Lnael5: AES1010.
175
8
273 ( )
Figure 5.4 Dendrogram of PFGE picture of fig. 5.3 :Lanel: isolate no. 30,
Lane2: AES81, Lane3: AES83, Lane4: AES91, Lane5: AES89, Lane6:
AES146, Lane7: AES182, Lane8: AES273, Lane9: AES284, LanelO:
AES287
300
250
200
150
100
Figure 5.5 PFGE of S 1 digestion of 14 isolates of P. aeruginosa.

Lanel: Marker, Lane2: AES81, Lane3: AES83, Lane4: AES89,
Lane5: AES91, Lane6: AES93, Lane7: AES146, Lane8:
AES 182, Lane9: AES273A, LanelO: AES284, Lanel 1: AES287,
Lanel2: AES934, Lanel3: AES988, Lanel4: AES998, Lnael5:
AES1010.
176
300 kb
250 kb
200 kb Chromosomal location
150 kb
100 kb
50 kb
Figure 5.6 Autorad of PFGE of fig. 5.5 after probing wth radio-labelled
blayim-2 encoding gene. Lanel: Marker, Lane2: AES81, Lane3: AES83,
Lane4: AES89, Lane5: AES91, Lane6: AES93, Lane7: AES146, Lane8:
AES 182, Lane9: AES273A, LanelO: AES284, Lanel 1: AES287,
Lane 12: AES934, Lanel3: AES988, Lanel4: AES998, Lanel5:
AES1010.
177
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 5.7 PFGE of Spe 1 digestion of 14 isolates of P.

aeruginosa. Lanel: Marker, Lane2: AES81, Lane3: AES83,
Lane4: AES89, Lane5: AES91, Lane6: AES93, Lane7: AES146,
Lane8: AES182, Lane9: AES273A, LanelO: AES284, Lanel 1:
AES287, Lane 12: AES934, Lanel3: AES988, Lanel4: AES998,
Lnael5: AES1010.
178
f^ H g l 1
W r:! i ■■:/:*
300 kb
250 kb , ............ ...... ..: ..^^ •- ^ l u i l l l l l l l
•
« *. 3t& &S3&J _ v 1
200 kb
150 kb
100 kb
50 kb
Figure 5.8 Autorad of PFGE of Spe 1 digestions from fig. 5.7 after
probing with radio-labelled blavm -2 gene. . Lanel: Marker, Lane2:
AES81, Lane3: AES83, Lane4: AES89, Lane5: AES91, Lane6: AES93,
Lane7: AES146, Lane8: AES182, Lane9: AES273A, LanelO: AES284,
Lanel 1: AES287, Lanel2: AES934, Lanel3: AES988, Lanel4: AES998,
Lnael5: AES1010.
179
5.2.7 Detection of class 1 integrons and transposons
Amplification o f class 1 integrons and transposons from clinical and non-
clinical P. aeruginosa isolates; AES30, AES81, AES83, AES89, AES91,
AES93, AES 146, AES182, AES273, AES284, AES287, AES934, AES988
and AES 1010 showed that 7 out of 15 yielded PCR products for class 1
integrons; moreover, the size o f the integrons amplified suggest that more than
one gene cassette is involved in each of the integrons. Specifically, PCR
amplicons integrons from P. aeruginosa isolates AES81, AES83, AES89 and
AES 182 (Table 5.2) revealed products of sizes 3kb, 2.5kb, 1.5kb and 1.5kb
respectively (Figure 5.9). The same integron was found in the two clonal P.
aeruginosa isolates; AES89 and AES 182 and composed of the gene cassette
sequence: intll, aadA6, ORF, qacEA/sull (Figures 5.10C&D) in spite of the
different site of collections of these isolates (Table 5.1). Class 1 integrons
from AES81 displayed the gene cassette sequence in tll, aadB, blaym-i,
dhfrAl aac6-Il, qacEA/sull) (Figure 5.10A). Isolate AES83 possessed the
gene cassette sequence in tll, aadB 1761, dfrAl, aac6-II and quacEA/sull
(Figure 5.1 OB). PCR on AES83 revealed the occurrence of the same genetic
context of the integron detected in AES 81 in addition to another novel
integron (Figure 5.1 OB).
1 80
3000 bp
2500 bp
2000 bp
1500 bp
m m>
1000 bp ihf
800 bp m
mm
600 bp
■mm
400 bp
200 bp
11 12 13 14 15 16 17
Figure 5.9 Amplification of class 1 integrons from P. aeruginosa

isolates. Lanel: Marker. Lane2: AES30. Lane3: AES81. Lane4: AES83.
Lane5: AES89. Lane6: AES91. Lane7: AES93. Lane8: AES 146. Lane9:
AES 182. LanelO: AES273A. Lanel 1: AES284. Lanel2: AES287.
Lanel3: AES934. Lanel4: AES988. Lanel5: AES998. Lanel6:
AES 1010. Lane 17: Marker
181
A
In tll
M
B
T T 7T T 777777T T T T rT T T ?
S /S /S /S /S S /S S S /S S S S j
M ir lllll
>*> *><>K
il>*><><><>1 i*>**!»*>*>*>*kl±*>* >
V S S //V //7 //////////S
T77TrTTTTrT7TTrT7TTrTr>
V / / / / / / / / / / / / / / / / / / / / / ,
in tii \(w tb£6yA K /m cR & 'fs?,
In tll :0a&A6::>: > _>L > > > > > > > > > >
i m -z m m s z i ,,,,,,, , , , ,,,
Figure 5.10 Genetic context of class 1 integrons found in Libyan P.

aeruginosa isolates. A: Class 1 integrons in AES81. B: Class 1 integrons in
AES83. C: Class 1 integrons in AES89. D: Class 1 integrons in. AES 182.
In tll: Integrase gene. aadB: gentamicin resistance gene.. blay\u-i- Carbapenem
resistance MBL gene. Aac6-II: aminoglycoside resistance gene, aadB 1761:
gentamicin resistance gene, dfrA: trimethoprim resistance gene, aadA6\
aminoglycoside resistance gene. QacEA/SuII: Quaternary ammonium
compound resistance gene and sulphonamides’ resistance gene.
182
5.3 Discussion
This section showed the resistance mechanism of some P. aeruginosa isolates
randomly collected from Tripoli and Benghazi hospitals, the isolates were
from clinical and non-clinical samples. Non-clinical samples were taken as
there is very little, if any, infection control in these hospitals and it was of
interest to see if isolates from environmental swabs matched those causing
infections. The Al-Jalla hospital showed the clonal incidence of multi-drug
resistant P. aeruginosa, these isolates exhibited the occurrence of different
class 1 integrons and in some MBL encoding genes.
The overall mechanism o f antibiotic resistance in P. aeruginosa utilises many
antibiotic resistance determinants (Walsh; 2005; Toleman et al., 2007), efflux
pumps (Morero et al., 2011; Cabot et al., 2011) and porin alterations (Muller
et al., 2011; Tomas et al., 2010). Specific antibiotic resistance determinants
can include MBL encoding genes and serine carbapenemases often linked to
mobile genetic elements, the former is exemplified by class 1 integrons
sometimes associated with Tn402-like transposons (Tato et al., 2010; Stokes
et al., 2006).
blayim-2 has been found as gene cassette carried by class 1 integron in P.
aeruginosa worldwide, in Poland (Toleman et al., 2003), Germany (Valenza et
al., 2010), Spain, (Rojo-Bezares et al., 2011), Venezuela (Guevara, A. et al.,
2009), United States of America (Aboufaycal et al., 2007) and Ireland (Walsh
183
& Rogers, 2008). It is also emerging Saudi Arabia (Guerin et al., 2005), Japan
(Yatsuyanagi et al., 2004), India and Russia (Toleman et al., 2007) Eastern
Europe (Bosnjak et al., 2011; Jovcic et al., 2011) and herein I report the first
detection of P. aeruginosa positive for blay\u-i in Libya.
Probing of PFGE gel of Spe 1 digestion with radio-labelled blawu-i indicated
that the two clonal isolates positive for the MBL gene had the same gene on
the same size DNA fragment. Nevertheless, analysis of the PFGE and
subsequent dendrogram demonstrated that the strains are not clonal and share
less than 85%. the results of this work are consistent with the studies of
Lagatolla et al., 2006 who described the incidence of high-level endemicity of
clonally related P. aeruginosa carrying v im - 2 - These finding are dissimilar
to the work of Nho et al., 2008 who reported the dissemination of genetically
unrelated isolates of P. aeruginosa carrying &/aVIM-2 in Korea. The results
are also conflicts somewhat with the findings of Aboufaycal et al., 2007 on the
emergence of different ribotypes of P. aeruginosa positive for blay\u-i genes.
Acquired class 1 integrons are considered major contributors of the multi
resistance phenotype expressed by bacteria due the capability of integrons to
capture gene cassettes and accommodate them within the variable region of
the integron by means of the integrase gene (int) and the site specific
recombination site (attl) and consequently an impressive gene array may result
from this fluid capturing machine from the gene pool surrounding the bacteria.
184
(Bennett, 2008). Gene cassettes include genes encoding functions such as
aminoglycoside (Lagatolla et al., 2006; Naas et al., 2006) and trimethoprim
modification (Hu et al., 2011). It also comprise major members of the class B
P-lactamases family (Jeong et al., 2009; Walsh et al., 2005; Castanheira et al.,
2004) and some members o f class A and D p-lactamases (Juan et al., 2009).
The detailed characterisation o f four Libyan isolates of P. aeruginosa
indicated the incidence of three different genetic contexts of class 1 integrons.
The bla\\u -2 positive isolates showed the occurrence of the same integron
structures composed of MBL encoding gene blay\u-i and two aminoglycoside
resistance genes; aadB and aac6-II genes. Similar findings were reported from
(Lagatolla et al., 2006; Rojo-Bezares et al., 2011). A novel class 1 integron
was detected in P. aeruginosa AES83, the integron contained two
aminoglycoside resistance genes and one trimethoprim resistance gene. It is
composed o f the genetic array aadB1761 and dfrAl and aac6-II. P.
aeruginosa isolates AES89 and AES 182 showed the incidence of the same
integron with exactly the same genetic context; aadA6 and ORF in the variable
region o f the integrons. The occurrence of this integron has been documented
in P. aeruginosa by several authors, Naas, et al., reported an ln5J class 1
integron composed o f aadA6 as a novel aminoglycoside adenylyltransferase
gene cassettes and an ORF which was the first description of the structure of
this variable region. (Naas et al., 1999). Similar findings were reported by
Shahcheraghi et al., who reported the incidence of 4 integrons with different
185
gene cassettes arrays acquired by 41 clinical isolates of MDR P. aeruginosa in
Tehran, Iran, one of the isolates contained a class 1 integron with a variable
region of aadA6 and ORF (Shahcheraghi et al., 2010). Some reports described
the incidence of this integron as part of complex genetic structure found in P.
aeruginosa (Nemec et al., 2010), other reports showed the occurrence of this
integron as part of complex structure (Naas et al., 2006). It seems that aadA6
and ORF containing integron first discovered in 1999 is the common ancestor
and is now found in different geographical areas such France, Iran and now
Libya.
Multi-resistant isolates P. aeruginosa has been found in different parts of
Libyan hospitals, it has been found in ICUs, Chest wards, patients or hospital
facilities, and even from the floors of some toilettes in the ICUs. Such
emergence of the resistant isolates is a worrisome subject and reveals the lack
of a proper hygiene and infection control programs currently operating in
Libya. blay\u-i was identified in one isolate of P. aeruginosa, that was
collected from stainless steel containers used to keep forceps and other
surgical tools in the Chest ward of Aljalla hospital , whereas the other isolate
was from a tip of catheter from patient admitted to the ICU of the same
hospital in Benghazi.
186
Chapter Six
Genetic & biochemical characterization

of a novel metallo-p-lactamase, TMB-1,
from a Achromobacter xylosoxidans
strain isolated from Tripoli, Libya
6.1 Introduction
The results in this section follow on from the determination of class 1
integrons in Achromobacter xylosoxidans (two integrons one at 3kb and one at
2.5kb), one in Stenotrophomonas maltophilia (2.5kb) and two isolates of
Citrobacter freundii each positive for a class integron of lkb. The isolates
were from non-clinical sources from the major hospitals in Tripoli, Libya.
Mobile MBL genes are becoming increasingly frequent and pose a significant
challenge to the treatment of Gram-negative infections world-wide such that most
MBL-producing organisms are only sensitive to colistin (Cornaglia et al., 2007).
These enzymes efficiently hydrolyze all P-lactams, including carbapenems (with
the exception of aztreonam), and are located on transferable genetic platforms;
namely, either ISCR elements or class 1 integrons. The class 1 integrons are
sometimes embedded in Tn27 or Tn402-like transposons (Tato et al., 2010).
However, several recently characterised MBL genes have been flanked or
associated with ISCR elements namely, blas?u-\ with ISCR4, 6 / o n d m -i with ISCR1
and blaA \M -\ with ISCR16 (Kumarasamy et al., 2010; Poirel et al., 2004; Toleman
et al., 2006).
Several different MBL-type enzymes have been described, among them NDM-1,
IMP and VIM derivatives being the most widespread (Bush, 2010). The blam?-\\te
(Senda et al., 1996) and blaviM-like (Cornaglia et al., 2000) genes have been
identified in clinically relevant bacteria belonging to the Enterobacteriaceae
188
family, in Pseudomonas spp., and in Acinetobacter spp. Whilst 6/ o n d m - i has
mainly been found in Enterobacteriaceae (Bush & Fisher, 2010; Kumarasamy et
al., 2010). Several other MBLs have been identified in specific geographical
locations, including SIM-1 from A. baumannii in Korea (Lee et al., 2005), KHM-
1 from C freundii in Japan (Sekiguchi et al., 2008), SPM-1 in Brazil (Picao et al.,
2009; Toleman et al., 2002), GIM-1 in Germany (Castanheira et al., 2004), and
AIM-1 in Australia (Walsh, unpublished data) were all identified in P.
aeruginosa. As hospitalized patients are subject to infections by Gram-negative
bacteria and, in Libya adherence to internationally accepted infection control
policies are not optimal, the hospital wards and immediate hospital environment
were examined for resistance to extended-spectrum cephalosporins. This study
reports these findings and further describes the genetic and biochemical
characterization of a novel MBL, TMB-1, from Tripoli, Libya.
189
6.2 Results
6.2.1 Analysis of samples from Tripoli hospitals
Thirty eight Gram-negative bacteria were able to grow on 10mg/l of
ceftazidime (Table D .l in appendix D). It lists the non-clinical swabs from
major hospitals in Tripoli, Libya. All swabs yielded isolates capable of
growing on 10mg/l o f ceftazidime. The results demonstrate that the
environmental isolates collected from the wider Tripoli environment and
hospital environment show a high level of resistance to third generation of
aminoglycosides and p-lactams. For example, one isolate of A. xylosoxidans,
AES301, displayed MICs of, 8mg/l, 2mg/l, 4mg/l, 16mg/l, 10mg/l, 32mg/l,
and 16mg/l to gentamicin, imipenem, meropenem, cefepime, ceftazidime,
cefotaxime, and aztreonam, respectively. Indeed AES301 was sensitive to
amikacin and ciprofloxacin (lmg/1) and colistin (0.5mg/l). All isolates grew
on media containing ceftazidime and were subsequently screened by the MBL
Etest strip to detect the presence o f MBL. AES301 gave a positive Etest MBL
result and together with the fact it possessed a class 1 integron, was
investigated further.
6.2.2 Genetic analysis of carbapenem resistance in A. xylosoxidans strain

AES301
All isolates were screened for class 1 integrons and mobile genetic elements
(Tn27, Tn402, and ISC7? elements) and 4 out of 38 isolates were positive for
class 1 integrons: one A. xylosoxidans (two integrons of one at 3kb and one at
190
2.5kb), one S. maltophilia (2.5kb) and two isolates of C. freundii each positive
for a class integron o f lkb. None of the isolates were positive for Tn27,
Tn402, and ISCK elements. Sequencing analysis of the class 1 integron PCR
products from A. xylosoxidans AES301, Lasergene package (DNAStar,
Madlison, WI) was used to study the nucleotide sequences and the deduced
amino acids. The nucleotide sequences were subsequently analysed
(http://www.ebi.ac.uk/Tools/sss/fasta/nucleotide.html). The two integrons
from A. xylosoxidans AES301 revealed two near identical integrons; the first
possessing the gene cassettes dhfrA4-aacA4-blaoxA-4 , and the second
integron-containing the gene cassettes blajuB-1-aacA4-blaoxA-4 (Figure 6.1)
(Appendix E). The carbapenem resistance could not be mated to either E. coli
DH5a or P. aeruginosa PA01 recipients suggesting the integrons are
chromosomally located. This inference was supported by Southern
hybridisation data using the 6/tfTMB-igene as a probe which back-blotted to the
A. xylosoxidans AES301 chromosome even though it possessed several
plasmids.
191
A 4 7 1 7 1 7 },
Iiitll dhfi'hA aacA 4 bIaOXA-4 qacAEisuIl
lilt I I blaTMB-1 aacA4 blaQXA4 qacAEistill
Figure 6.1 Genetic context of two class 1 integrons found in A. xylosoxidans
AES301 and the primers used to sequence the structures. A. Class 1 integron
consisting of the gene cassettes: dhfr A4 gene, aacA4 gene, blaoxAA and the
qacEA/sull fusion. B. Class 1 integron consisting of the gene cassettes: blarm b - i,
aacA4, blaoxA-4 and qacEA/sull genes. The white ellipse represents the hybrid
promoter from I n tll . The black ellipse represents the 59bp elements at the start of
each gene cassette.
6.2.3 Cloning and transconjugation experiments
The results o f cloning o f the class 1 integron into E. coli DH5a produced 3
types o f colonies when 50pi o f the broth culture was streaked onto L.B. agar
plates supplemented with 50 mg/1 of Kanamycin, X-galactose and IPTG;
white colonies, white colonies with blue spot in the middle and dark blue
colonies. Amplification o f the class 1 integron from these colonies showed
that some white colonies and dark blue colonies produced 2kb and 4kb PCR
products which were different from the expected size of class 1 integron used
in the cloning experiments. Sequencing of these PCR products did not give
192
any readable sequences revealing the miss-priming of the oligonucleotides.
These results did not produce any cells positive for &/atmb-i- The results of
transconjugation experiments showed that the GFP E. coli was not able to
grow on L.B agar supplemented with 50 mg/1 of rifampicin and 4 mg/1 of
cefotaxime revealing that the 6/atmb-i failed to transfer to the GFP E. coli and
thus is probably chromosomally mediated.
6.2.4 Genomic location of A / a t m b - i
The results of PFGE separation of S 1 digestion of the whole genomic DNA of
A. xylosoxidans and selected environmental isolates is shown in figure 6.2A.
Probing o f the PFGE gel of SI digested genomic DNA with radio-labelled
/j/ajmb-i is illustrated in (Figure 6.2B). These results showed that blajuB-\ is
located on the chromosome. These results are also in accordance with the
cloning and transconjugation experiments that failed to detect any movement
of blaj m b - i from parents to recipients predicting the chromosomal location of
this 6/atmb-i- The results also showed an additional TMB-1 positive isolate of
A. xylosoxidans which was found in the ICU male surgery ward in Tripoli
central hospital.
6.2.5 Comparison of TMB-1 with other MBLs
blajuB-x contains 735 nucleotides encoding a protein of 245 amino acids and
possessing all the key motifs of Ambler class B p-lactamase, SDS gel
electrophoresis showed an approximate molecular mass of 25 KDa. At amino
acid level, TMB-1 was most closely related to DIM-1 (62%) and GIM-1
193
(51%), and showed only 48%, 31%, and 29% identity to IMP-1, VIM-2, and
NDM-1, respectively (Figure 6.3) (Koh et al., 2004; Poirel et al., 2010;
Castanheira et al., 2004; Yong et al., 2009; Garcia-Saez et al., 2008). TMB-1
also possesses virtually the same key residues as DIM-1 that make up the zinc
binding residues and the secondary residues supporting the active sites
including the putative loop used to facilitating binding of (3-lactams during
hydrolysis (Figure 6.4). The secondary structural comparison of TMB-1 with
VIM-2, (Garcia-Saez et al., 2008) shows that TMB-1 possesses the key zinc
binding residues for B1 MBLs; H isll6, H isll8, and Hisl96 (zinc 1) and
Asp 120, Cys221, and His263 (zinc 2) (Figure 6.4).
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
Figure 6.2 Detection o f genetic location o f blaTMB.\ in A. xylosoxidans. A.

PFGE o f 51 digested DNA. Lanel: AES301. Lane2: AES302. Lane3:
AES303. Lnae4: AES304. Lane5: AES305. Lane6: AES306. Lane7:
AES307. Lane8: AES309. Lane9: Marker. B. Autorad after probing with a
radio-labelled blaTM b - i o f PFGE gel from fig. 6.2A.
194
The most noticeable difference between TMB-1 and VIM-2 is a gap in the N-
terminus o f the TMB-1 protein just before the beginning of the first (3-sheet
(pi, Figure 6.5). This gap in TMB-1 is situated just prior to the “flapping
loop” o f VIM-2, (Garcia-Saez et al., 2008) further, there are several amino
acid differences in this region; namely, (VIM-2 to TMB-1) Q60S, S61R,
F62V, D63E, A66G, V67L, and a gap at position 65. This region is also
diverse between VIM-2 and VIM-7 where it has been suggested that this
contributes to a more flexible “flapping loop” (Borra et al., 2011).
Interestingly, DIM-1 possesses the same sequence as TMB-1 in this region
with the exception o f the gap and the amino acid changes N63E and F65W
(DIM-1 to TMB-1) (Poirel et al., 2010). An additional gap in TMB-1 between
p7 and P8 compared to VIM-2 is also observed (Garcia-Saez et al., 2008)
(Figure. 6.5).
195
TMB-1
- DIM-1
— GIM-1
IMP-1
SIM-1
KHM-1
SFB-1
SLB-1
- NDM
VIM-1
IND-1
JOHN-1
SPM-1
164.6
1 1 1---------
160 140 120 100 80 60 40 20

Amino Acid Substitution per 100 resid u es
Figure 6.3 Dendrogram of Comparison of amino acid sequence of the P-lactamase TMB-1 and those of other
acquired MBLs (DIM-1, GIM-1, IMP-1, KHM-1. NDM-1, VIM-1, SPM-1 and SIM-1) and several naturally
occurring MBLs (IND-1 from Chryseobacterium indologenes; JOHN-1 from Flavobacterium johnsoniae;
SLB-1 from Shewanella livingstonensis; and SFB-1 from Shewanella figidimarina) (Koh et al., 2004; Poirel
et al., 2010; Castanaheira et al., 2004; Sekiguchi et al., 2008; Yong et al., 2009; Lee et al., 2005; Toleman et
al., 2002; Tato et al., 2010; Naas et al.,2003; Poirel et al., 2005; Lin et al., 2005
196
TMB-1 - M R'P - P L F U I I P I f i K P - AFftNKKIPG IoKVK BlDNQVFLHirSYGRVBGIfaLVGSNGLVVISQQKAPI IDTPMSESDTKKIiYDM IRSKK YELAG&16T
D IM -1 N RT H FT A tiL L L P K L fi CIJUIE«BVPK 1*311 tt KVKEN1 F1.HTSYS HVNG>GtATS SNG L,W I" j KQNAFIVDTPN CDRDTETLVl INTI It KNG YELLGSTt'ST
G IM -1 - WCNVLV V & X X . LVA.Ij P - - WJkOrJHK.P --------------- DBTVI -K_1£ IX3VYLBTKKKN IBGTO HiVDBNQLW I.D (fNJA Y IID T PNfiKnDTlIJjl-GIIATDRG -YO’^ A S I S T
IM P -1 - M fitL S V rp J P L P C S I - AT-JUUKSI^PD-------------------U I E KIDEaVWffnBPEEVNGWaWPKHaLWl.VNAKAYLIDTpvTAKDTBILVTIIPVEHa YKI KGGIGE
I3CM-1 -------------- - N K IA L V I CfG LLIiFT N M - VCUDDEUPH - - I D I O - CINDaVYLYTATfEK I B G M a L V G C N aL W L D N IN H Y E IB T P I KATDTEHLVJCWIDAQG - P T A K A C IG T
BTOM- 1 MBLPN IM KPVAKLETALAAALM LCGCM PaBI R PT I FTGDQR FGDLVFH-QLAPNVWijH TCY^DM PG? <jAVA ENG I, a V P V I , VVDXVVMTDDQTAQ I I..Nit I K Q E IN L P\rftLAV’J T
SIM - 1 U R T L L IL C L P Q T L N T - - AFJU*BEAOITC» feKXSE U K K Q I YCaSTTV?FQ E YX jjFO IV3C JCQClXiWI*D N'HKAY I-1D TPA CAtlDTIS N.I>VN1I I »K ICND P T V N Q C IS T
F. PM 1 - - KNEPKSPALIyGFM GAi'CLLLVAGAPLEAJCEEDHVDl.PYN LTA T fClO ^ D V I ^ n r D R D P T S E - M^LVAKMLD G T W l V E E E N L GirQTXJKCHfl** AKTM K P m 'V A D r T
V IM - 1 - M LKVICCLLVYM TAEVM AVAEPLAHEGBPEflffyPTVNK I PVG.EVRLY - QlADCJVWEHl AT<}SFD© AVY P S N Q til W Z*CKDKDD^3tJDTJcMGA KNTVUXXÂ.KXK K .Q I O LPVT RAVBT
I N D -1 M3CK - E I R P P 1 V 6 I L L S P ------------ÂSAOVKD FV I K P PIKMMluSC 1 YHtTFGVPGii* - K EY «AJI£M YLVTKXGW L?tJVT*fEK I OYOSWOJrriKKHHNL P W A V F A T
<IOHltf~l MBtKIiAE I ILPLA A VSN G - - LOOGKNEP JjOXS - HLTGDPYVYRT FN DY KJQt T K IJU a A M Y V iT D K aW L F B A P irD K T O F O P L ix O E I KA«3^ NK3KW M LFO|
E F B -1 I I I E A PErA H E N E O Q TD gSN T D A V K A PQ O Q PT KKFL.S - P L V P D V Y ta o S Y X Q V S ^ ^ I^ K E N G liV V V Q N K Q sA P I ID T P N T D e O T /ilL V D ir ETQQG - L T \ n : A £ I S T
SDB- 1 1 IL S L P G Y S H H V E P T S - - T T 3 G S V T S S L K Q -QJUSIS KLAO«JVYI^miSYXlW£M FtSHUVE ANQLVVI KDKQAFI ID T P ^ D N D T O K L V tM Z T Q Q G - F I P V A S I S T
1YS 118 *30 2-,^
TMB-1 H EKED K.TAG I KWEHGKE 1 TTYASJAI/Tilttri^KJlM KK Q A R £K F K G N K 7--------------------------------------------------------------------------------SLMDQPJUKVYYPGGGHT tD N L W M I P £ S 1 ILY O G C P IR E L E S S G U 3
D IM -1 h w k k d r t a g i k m x js td g e X £ t y a j t t c t m h l ^ w c e n k k m - p jik y tljc g n k .' TX,vi>ai, iB V r Yp g o g h t i c h w v w x > p k s k i l f g g c fv te i s l d s e g 1 x 3
GIM -1 MEHfiei>PTAGi:*CI.£JSr£:AJS:il>YYTBKl.TKAIJLJA.aSt>aKP - V P T H YWKnOKP -------------- --------------------TLC NClt>1 8 L YY PG AG HT FilDN I VA%f: 11* KC3GC1. MISIiEWKGLQ
IM P -1 H P K E O C T C ^ I KWLRC RC1 ITYAGKLTMEIXKY L:GtV QATNEPbGVNY • ...................... MLVKNJCI 8 V F Y P G P G H T P O N V W r^ P E R K IL T O O C P IK PY GLD
xsm-i H i^rm E T C X » rA PX JrE lt« ir> rY A S K l*T I»O U l,K N JCOES - O A T H B F G M IP Y ------------ - ............- - -MCXjKKIC I lA r Y P G A G H T PD N L W M L PK Q B C IL P tiaC F ’.'K PE O tG
NOM- 1 HJVKQDKMCK3Mn/aiHA.AGlATY«MAI^SJICLAPC*W3MV - - AAOHQLTFAAMGM V E P A T A P N P G I P t ^ r 't P G PGHT SDN I T V G jD G T D lA FOGCIÎKDE KAKEt^Q
S IM -1 H ('H D D C T A G I K M L N T ICE I iT Y A G K . L T N E l .U S ' ? : N a K T ------ Q A K H S P D K E E P ---------------------- --------------------------- W LV K N IC I t t I F Y IK S I G H T Q D N E W W I PM KJC11* F Q G C P I K PH QLG
S PM -1 H PKIUOTOGW B I Y3CKMGA KTM S SI J LT KQl. RL S » i KKD R I KAABFYIMHD1<KPR I L S SHPVPADN VFD LK OG K VPfiPEN ELVRV SFPG PA H E PITOVWY F3PK:.KJ£L£* F<3C»CMXKPX EJW3
V IM -1 HPKC-DRVOG’'; DVLRAAGVATYASP ETS RI..AEA8GN8 - I P T H fiL B G l.S E ------------ EQDAVRFQPVEIj FYPGAAttET'DHSrt.WYVP'GAN VX*YG«CA V H E L efiT S & &
I N D -1 HEKDDRA GDLEP FKM105X XTYAXAKT WKKjkJUCc a KA-------T f i T H I l l r a K P -----------------------------------------------------------------------------Y R IG G EE FW D FI«G EG H T AIIWVVVWF1FK YMVl*LK3MSC!I-<VK2S3YSATDfjQ
JO H N -1 H S K B U R A ilG P D F Y IIC IG I XTYS I KLTDDILKX N KK P RAE F 11 SPTDTT------------------------------------ ----------------------------------P T V G N K T F W Y tP G K G H A P lW 1VANFi: KEK ILY GG CPV KSAEALDLG
S F B -1 HJSHQDRAtôraYI,aSIEQOXATTtVBmCTORUUTANKLS-------TA EH TFRTK Q K .......................................................................................................ThOQQI^ I KVYPLOAGHHT/imi^LVWI^PKQQXI*F G Q Q ^ I K g L S S B T U g
GLB - 1 g « |^ j j R A Y lij^ Q O X W T V e E T T 0 0 3 ti* T E N D I T ---------T3*XjrTFTl3MQ Y -------------- | | |
363
TMB-1 YTGRAKI DOM PQS ARNT1EK Y PEA I I W PQHG K IO D PELLKHTICV LAP KAE WKANHG DR
D IM -1 YTQRUA12XDOMCS SiAQM/c-.BRYi- K A Q IV I POKGKIGD I AkJLJCSrr I*A12TABKTK.C I QPNANAEAD
-GIM - 1 Y /G D A S I E E tA D f i I K» TVER KYPI Q M W PQ 8 G r / a £ E D I L D B t I DLAE EAENK LMQFTAEAEAD
IMP - 1 Jl LCDAN1 HAM PiOSJlKI, L.K.GAYG X A E l.W P E K E EYGOA S XJUHX.YX.BQAV.EG7-JBTK£ KR PE X PEN
MUM- 1 N l*£l H AV I AEW PAEAE I C L I A R tS K A T M W P G K G ^ D A E tl* K K tR Q R A V EAI-AA KJC
NEW - 1 NU3DADTEH Y AA SAH A FaAA PPlAC M IVMEHEAPDSR AA I THTAR M&IJK.I.P
S IM -1 N L»£ DA-NI, EAMPGSAK KMIC EYE XAICl.V I P £ « £ KIG DA E I1I4JC-TN HC*A1ICGIME E K E I P P L IN
SPM -1 Y I j IDANV (CAMPDKABlR1<JCX - - F D A O V I PaHGENOGPRM V N IT T KVAEKAV 3KMRL
V IM -1 N VADAD l.A EU PTfiV K Rl Q KHTPEAK W I P < H « G L L C - L tC 'H T A N VYKAHKNR£ VAE
IBRD-1 Y I KJ&ANV P-OMPKTINKTLX AJCYfi1ATL I 1 PQKI3 EM KGEE.H V KfcfTl.KIsl^lKK
JOHN- 1 YU3DADVKENgKB I KKVQARFKKPDY I I EQHpDDTtTE KB£1*KTXTI.KD\T3KYI.AQ K£ AGK.K
S P B -1 YTQEADL K<J« P L T VA ICVÂQPICAKI W P Q K G X XOUTE I*L £ H T I ULZ^TQ
GlaB- 1 YTaKADLOQW PI*T IA KVQ AQ FFQV K.I W P Q H G < :vaD K A ^LK H T I E t i - I PKN ETVBfE E E
Figure 6.4 Comparison of amino acid sequence of the p-lactamase TMB-1 and those of other acquired MBLs (DIM-
1, GIM-1, IMP-1, KMH-1, NDM-1, VIM-1, SPM-1 and SIM-1) and several naturally occurring MBLs (IND-1 from
Chryseobacteriumindologenes; JOHN-1 from Flavobacteriumjohnsoniae; SLB-1 from Shewanellalivingstonensis;
and SFB-1 from Shewanellafigidimarina) (Koh et al., 2004; Poirel et al., 2010; Castanaheira et al., 2004; Sekiguchi
et al., 2008; Yong et al., 2009; Lee et al., 2005; Toleman et al., 2002; Tato et al., 2010; Naas e/ al.,2003; Poirel ef a/.,
2005; Lin e / «/., 2005). Shaded amino acids are those conserved with TMB-1. p-Lactamase numbering was according
to the BBL nomenclature (Galleni et al., 2001). . Q„
. T T---------- 2.
V IH
TMU 1 2 a rj? E L L s # : v Y : ;: a s E K f J i a s : l a p s v l s s - b y p t v i f - W O v c e I V n i l y O H a dBm w s f f i i 0 i f - s f : R . a I v lv rR
1 Crr.F c ■ - ........................ f- -r t t a . - o [ L i ^ 3 3 y |E - e ® H r i f i |> ^ j v t i i < \r?BM,cftjvs§j
VZM 2 P a2 «3 3fi
<?; •;a ;oi •;•
V IM -2 70 i.trJ^ -E^ lG-'kl - □ E E - ' E - - -;U SLifTuM-. -Bffli-'HLiBlK v gHvl vB maa^'--EjEEB-- -:E
T w a a -i l i C E E -E i : E j' k a FT^ " )•: •> S c k H - J i : : m * ! . k y 3 | l a i o S O E , i l . F ! 0 r -A01 • :••) i ( 3 B B 0 a i | 3
oc4 &s do (3 1 0
TT ----------► T T ---------- 1
V T M -2 140 P .F ' L A E VV B E n B 1 ? ffH 0 L 3 B E .1 5 S Q = 3 A p ? . ? E - * /E £ F f l ^ A A g I i f :,Vi y

TMB 1 120 N iii L K r EEMB^ ah ■0 f . -iLHr.Eh UB-. OGcl'.v.i-l ®0BSw'■S l i b u H S f l E a
VZW-2
o5 (ill «6
TT
V T O -2 210 W V A D g D ‘ A j ' E O T E I I F P. : ] g l j l T E H Q g d F E - E B B E L P f i j C L B m g B B B T l I I V V l K A l - l E Q n - ~ 'J . . 'A R
tm b - 1 167 v t rr r?Qr-r t n ~m : g ' , p ' f ' T| n - ' K C B Q n x j t B v T O I F v t g j n - k v | t . a |k k s | . s , \ " n r , r . | p
Figure 6.5 Secondary structure of TMB-1 compared to that of VIM-2 (Garcia-Saez et al., 2008).
The (3- strands and (3-helixes are indicated above the TMB-1 sequence. The conserved residues
are indicated in black. The conservative amino acid substitutions are boxed. The figure was
obtained with ESPript software (http://espript.ibcp.fr/ESPript/ESPript/).
198
6.2.6 Kinetic properties of TMB-1.
The kinetic properties o f TMB-1 were compared with that of DIM-1 and GIM-
1 (Table 6.1) and were broadly similar with the exception for the rate of
turnover o f substrates (Kcat values) (Table 6.1). The Km values for TMB-1
were similar to DIM-1 and GIM-1 for the penicillins and cephalosporins but
were higher for meropenem indicating that meropenem is not a “natural”
substrate for TMB-1. The Kcat values for TMB-1 were similar for the
pencillins compared to GIM-1 but were significantly less (20 to 500-fold) than
both DIM-1 and GIM-1 for cefoxitin, cefuroxime and ceftazidime (Poirel et
al., 2010; Castanheira et al., 2004) (Table 6.1). TMB-1 also possessed lower
Kcat values for the carbapenems (3 to 30-fold) compared to DIM-1 and GIM-
1. These data further showed that the efficiency of the enzyme {Kcat/Km) was
significantly lower for the cephalosporins and carbapenems (Table 6.2). Such
differences in kinetic values is interesting given that TMB-1 and DIM-1 are
similar and that their sequence over the “VIM-2 flapping loop” is nearly
identical, further suggesting that the reasons for these kinetic differences could
lie elsewhere in the TMB-1 structure (Figure 6.5).
199
Table 6.1 Steady-state kinetic constants o f TMB-1, DIM-1 and GIM-1 (P oirel et al., 2 0 1 0 ; Castanheira et al., 2 0 0 4 )
Compound TMB-1 D IM -T G IM -1b
k cat Km kCM/ K m kc-M K,„ kcm / A,„ kcM Km kQM/ K m
(s'1) (l-iM) (s‘7|iM ) (s'1) (HM) (s'VpM) (s'1) (pM) (s'V^M)
Ampicillin 3.3 27 0.122 20 110 0.182 3.3 20 0.165
Piperacillin 3.3 72 0.046 NR NR NR 6.9 69 0.1
Cefoxitin 0.3 69 0.004 8 20 0.4 8.3 206 0.04
Cefuroxime 0.1 9 0.011 NR NR NR 5.9 7 0.843
Ceftazidime 0.07 31 0.002 3 50 0.06 18 31 0.58
Ertapenem 0.4 31 0.013 NR NR NR NR NR NR
Imipenem 1.7 200 0.009 35 80 0.438 27 287 0.094
Meropenem 1.4 75 0.019 50 10 5 2.7 25 0.108
Aztreonam < 0.01 ND ND < 0.01 ND ND ND ND ND
“Poirel e t a l . , 2010
bCastanheira et a l .,2004
cNR: Not Reported
dND: Not Detected
200
6.3 Discussion
Non-clinical isolates collected from the major Tripoli hospitals were able to
grow on media containing ceftazidime. MDR Gram-negative bacteria e.g A.
xylosoxidans, P. aeruginosa, A. baumannii, E. cloacae and C. freundii were
detected in the hospital environment inside and around the hospitals. The
occurrence of MDR strains in the clinical setting of Tripoli hospitals reveals
the lack o f hospital hygiene in these hospitals.
Investigation into the incidence of antibiotic resistance genes embedded in
class 1 integrons was surprising. Two class 1 integrons were detected in A.
xylo so x id a n s , the 3kb integron had a novel MBL gene (& /« t m b -i) in the first
position followed by two antibiotic resistance genes aacA4 and blaoxA-4 ,
whereas the 2kb composed of dhfrA4, aacA4 and blaoxA-4- The occurrence of
such integrons is unusual in terms o f their genetic context and more
importantly the discovery of a novel MBL in a non-clinical isolate because of
the rarity o f environmental MBLs whose genes are shown to be mobile.
The occurrence of A. xylosoxidans in the non-clinical settings of Tripoli
hospitals may perhaps indicate the dissemination of 6/<2tmb-i in Tripoli and
across Libya. The occurrence o f blaju^-x in an environmental strain may raise
the question o f whether MBLs are originated from environmental bacteria as
201
the case of origin of blacix-u-3, form Kluyvera ascorbata proposed by
(Rodriquez et al., 2004).
TMB-1 has all the key motifs of Ambler class B p-lactamases; it shares 62%
similarity with DIM-1 (Poirel et al., 2010) and 51% with GIM-1 (Castanheira
et al., 2004). TMB-1 and DIM share the same key residues that facilitate
binding o f these enzymes to P-lactam antibiotics during hydrolysis. Secondary
structure of TMB-1 showed that these enzymes posses all the key zinc binding
residues (Hisl 16, Hisl 18 and His 196) required for zincl activity and (Asp 120
and His263 for zinc 2 activities) as reported for all class B1 MBLs (Osano et
al., 1994; Lauretti et al., 1999; Toleman et al., 2002; Castanheira et al., 2004;
Lee et al., 2005; Gupta, 2008; Sekiguchi et al., 2008; Yong et al., 2009; Poirel
et al., 2010).
Achromobacter is not a key pathogen although a growing number reports
indicate that it is capable o f causing UTIs (Tena et al., 2008), ocular infections
(Reddy et al., 2009), contamination of dialysis (Turgutalp et al., 2011) and
ultrasound equipment (Olshtain-Pops et al., 2011) and can cause additional
complications in cystic fibrosis patients (Lambiase et al., 2011; Ridderberg et
al., 2011). Interestingly, although AES301 carrying TMB-1 was found from a
ward surface swab, the same strain could not be identified from a clinical
source although in Libya clinical diagnostic microbiology may not normally
scrutinize strains to species level. To date only two cases of MBL genes (both
202
bla\i\M-i) have been reported from Achromobacter spp. - from Greece
(Soflanou et al., 2005) and Korea (Shin et al., 2005) and both carried in class
1 integrons. All other MBLs discovered IMP; VIM; SPM-1; GIM-1; SIM-1;
AIM; KHM-1; NDM-1 and DIM-1 were detected in clinical isolates from
patients suffered from serious infections. (Osano et al., 1994; Lauretti et al.,
1999; Toleman et al., 2002; Castanheira et al., 2004; Lee et al., 2005; Gupta,
2008; Sekiguchi et al., 2008; Yong et al., 2009; Poirel et al., 2010).
This is the first MBL reported from Libya and being a new MBL subclass B 1
provides further evidence of the structural heterogeneity o f this group of p-
lactamases.
203
Chapter Seven
General Discussion
This study investigated the mechanism of antibiotic resistance in randomly
collected isolates of Gram-negative bacteria in Tripoli and Benghazi (Figure
7.1). The isolates were from clinical samples recovered from patients admitted
to the hospitals and from the hospital environment and for the purpose of my
thesis these are regarded as non-clinical samples. These swabs were from
floors, walls, bedsides, toilets, workstation tables, mechanical ventilators,
oxygen suppliers, stainless steel containers, curtains, baby incubators, trolleys
and other instruments used in the hospitals in particular ICUs. The non
hospital environmental isolates were swabs collected from streets in Tripoli
and Benghazi; the samples were from floors and dusty areas in the streets.
1
M e d i t e r r a n e a n S e a : Crete
.TUNISl (GREECE)
'RiPOLI (amah
Kftums
Figure 7.1 Map of Libya f ! Mi^fStah G a ff o f
-J o b r u k
J Zuwarah Siam
showing the important .Ghadamis Ra's
Lanuf
'Marsa al
Burayqah
cities and their locations
(http://www.google.co.uk/imgres?q=li
byan+map&hl=en&gbv= 2&tbm=isch
ALGERIA
NIGER
0 150 300 fern CHAD i SUDAN

0 ISO 300 mi I
The data in my thesis clearly demonstrates the emergence of MDR Gram-
negative bacteria in hospital settings that include clinical and non-clinical
isolates. In total, 171 isolates were recovered for this study.
205
Enterobacteriaceae represent the most numerous of the Gram-negative with K.
pneumoniae as the most frequently isolated bacteria. High prevalence of
ESBLs was detected among K. pneumoniae and E. coli, more importantly
blacTx-M group 1 were found widely disseminated in association with ISEcpl
being located immediately upstream of most of the ESBL encoding gene.
The highest incidence o f blacrx-u group 1 was detected among clinical and
non-clinical isolates collected from hospitals. K. pneumoniae and E. coli
isolates tested for the occurrence of conjugative plasmids responsible for the
movement o f blacix-w group 1/lSEcpl. The study showed that these plasmids
can move blacxx-u-xsftSEcpl genes from the resistant isolates to sensitive E.
coli, leading to E. coli transconjugants expressing an MDR phenotype. It is
worth mentioning that plasmid mediated blacix-u -\5 associated with ISEcpl
has been detected in an environmental isolate of K. pneumoniae AES817
(STS 11) cultured from one of Benghazi streets - this isolate clearly has not
been recently exposed to antibiotics. These findings are an alarmingly
indication that an outbreak of MDR K. pneumoniae and E. coli isolates can
occur in different hospitals that provide different services to patients; these
hospitals are maternity hospitals, pediatric hospitals, surgical hospitals and
general hospital in Tripoli and Benghazi.
The occurrence of K. pneumoniae and E. coli in Libyan hospitals is interesting
in terms of the prevalence of clonally and non-clonally related 6 / « c t x -m - i 5
206
positive isolates (Lee et al., 2011; Webster et al., 2011; Alfaresi et al., 2011;
Fam et al., 2011; Al Sweih et al., 2011). The collection of isolates being
clinical, non-clinical or environmental was non-representative seeking the
occurrence of any resistance mechanism in these isolates. Among all the
isolates positive for blaax-u group 1, 4 pairs of K. pneumoniae isolates were
found clonally related. One pair represented two isolates that were found in
two different hospitals in Benghazi - isolates AES 135 and AES 140 that were
cultured from urine and blood samples, respectively.
The occurrence of several clonally related K. pneumoniae and E. coli that
resulted from the non-representative sample collection reflected the total lack
of appropriate infection control programs in Libyan hospitals. Lack of hospital
hygiene is another reason that has contributed to the emergence and spread of
multi-drug resistant isolates of K. pneumoniae and E. coli in Libyan hospitals.
The overuse of extended- spectrum cephalosporins to treat infections in Libya
may be another significant factor for the appearance of clonally and non-
clonally related isolates of K. pneumoniae and E. coli in Libya. (Ito &
Kamimura, 2011; Wang et al., 2011)
The occurrence of blacix-u group 1/lSEcplon different plasmid sizes in K.
pneumoniae and E. coli isolates suggest the movement of these genes by
conjugative plasmids (Lavollay, et al., 2006) as shown from data on mating
studies in a subset of these isolates. The ability of these isolates to mobilize
207
and facilitate the spread of antibiotic resistance genes may explain the
frequency of blacTx-M group MlSEcpl in non-clonally related species of K.
pneumoniae and E. coli (Woodford et al., 2004; Gonullu et al., 2008;
Lavollay et al., 2006, Yu & K. Cheng, 2004; Ramdani-Bouguessa, et al.,
2006; Abbassi et al., 2008). A possible explanation for the different plasmid
location of blact x -m group 1 is due to the presence of the insertion sequence,
ISEcpl that is known to move and promote the expression of the ESBL gene.
Two suggested mechanisms of blacix-u group 1 acquisition are proposed: the
movement of blacix-u group 1 by conjugative plasmids and the role of the
insertion sequence IS Ecpl in mobilizing blacix-u group 1 within the strains
(Abbassi et al., 2008; Poirel et al., 2003; Naseer & Sundsijord, 2011; Younes
et al., 2011; Gonullu et al., 2008; Villa et al., 2010; Partridge et al., 2011).
Data on Libyan E. coli give evidence of the occurrence of both mechanisms in
the movement of antibiotic resistance determinants for two reasons, blacix-u
group 1 and ISEcpl were detected on two different plasmid locations in parent
and recipients which suggests the role of IS Ecpl in mobilizing blacix-u
groupl (Rejiba et al., 2011; Smet et al., 2010) to a different size plasmid in
recipient. An alternative suggestion is the creation of a co-integrative plasmid
during conjugation. Similar resistance profiles were detected in parents of K.
pneumoniae and the E. coli transconjugants (GFP E. coli and E. coli J53)
suggesting that many of the antibiotic resistance determinants expressed by
208
the Libyan isolates o f K. pneumoniae and E. coli are located on conjugative
plasmids (M nif et al., 2010; Cullik et al., 2010; Partridge et al., 2011).
In addition to previously reported K. pneumoniae clones known to carry
blact x - m -15 e.g. ST15, ST29, ST101 and ST147, this study provided new
MLST groups - K. pneumoniae ST509 and ST486 and, additionally, a novel
environmental allele ST511 was found carrying 6 /<2 c t x - m - i 5 on different
plasmid sizes. The results of MLST data provided further evidence of the
incidence o f blacix-u -\5 in different clones of K. pneumoniae and also shows
novel sequence types are involved in the carriage of 6 /« c tx -m -i5 in Libya
(Nielsen et al., 2011; Pitart et al., 2011; Papagiannitsis et al., 2011; Hrabak et
al., 2009; Damjanova et al., 2008).
RAPD and MLST techniques used in this study show that they are similar in
detecting the strain relatedness between K. pneumoniae isolates; however,
they still are less discriminatory than PFGE which is based on genomic DNA
separation rather than amplification of specific fragments or housekeeping
genes. This study supports the application of RAPD technique to correlate the
relation of bacterial species to each other but not in determining the detailed
clonality within a species. Unsurprisingly, RAPD and MLST when compared
with PFGE demonstrated quite different results - some isolates appeared very
similar by RAPD but distinctly different with PFGE. Other isolates e.g. K.
pneumoniae AES74, AES59 and AES 1029 that belong to ST15 were
209
comparable by RAPD and MLST, but by PFGE demonstrated a low-level of
similarity. However, RAPD may help in the general assessment to determine
the broad similarity among bacterial species and it is rapid and inexpensive.
The MLST method is reliable but depends upon sequence stability among
housekeeping genes. PFGE can provide very detailed information on the
differences between species or subspecies; but it is specialized, temperamental
and quite expensive compared to MLST (Hotchkiss et al., 2011).
Class 1 integrons, whether alone or in association with Tn402-type or Tn27
transposons are among the most represented mobile genetic elements
responsible for capturing genes in the form of gene cassettes. Among the
Libyan isolates tested, 11 different type of class 1 integrons were detected in
21 isolates of Enterobacteriaceae and non-fermenters. Twelve class 1
integrons were detected in K. pneumoniae, 3 in E. coli, 3 in P. aeruginosa and
2 in A. xylosoxidans. Most integrons detected in this study carried
trimethoprim and aminoglycoside resistance genes which are typically found
as gene cassettes. Six different integrons were found in K. pneumoniae
isolates, two of which were embedded on Tn402-type transposons, of these 6
integrons, 5 integrons have been previously reported from different
geographical areas. One of these integrons {Inti, dfrA17, aadA5, qacEA) was
also found in K. pneumoniae and E. coli isolates examined in this study
(Vinue et al., 2008; Tang et al., 2011). Another integron harbouring dfrAYl
and aadA2 was detected in K. pneumoniae clinical isolate AES48. This isolate
210
was identified in this study as ST 147 which is known as a world wide ST
contributing to the emergence of 6 / « c t x - m -15 genes. The genetic context of this
integron has been reported in clinical isolates of E. coli in Asia and Europe
(Yu et al., 2004; Tang et al., 2001; Saenz et al., 2009; Vinue et al., 2008)
These findings show that the same resistance mechanisms are disseminated
worldwide, and also show that the Libyan isolates share the same genetic pool
with bacterial species worldwide.
Class 1 integrons detected in non-fermenters included in addition to
trimethoprim and aminoglycoside resistance genes, antibiotic resistance genes
responsible for conferring resistance to broad-spectrum P-lactams. blawu-i
was detected in two clonal isolates of P. aeruginosa collected from different
places in the same hospital, AES81 was non-clinical sample found in a
stainless steel container and AES 83 was recovered from a clinical sample
cultured from a tip of a catheter. These findings show the potential of P.
aeruginosa to acquire class 1 integrons in Libya and that it is widespread both
in clinical and environmental isolates as have been reported worldwide
(Valenza et al., 2010; Rojo-Bezares et al., 2011; Guevara, A. et al., 2009; Van
der Bij et al., 2011; Piyakul et al., 2011).
Perhaps the most surprising finding from this study was 6 /a tm b -i, a novel
MBL gene discovered in A. xylosoxidans cultured from Tripoli central
211
hospital. blajMB-i has been detected as a gene cassette embedded in the first
position of class 1 integron followed by an aminoglycoside resistance gene
(aacA4) and oxacillinases gene (blaoxA- )- The

4 same A. xylosoxidans isolate
possessed a second near-identical integron composed of aacA4, blaoxA -4 but
with blajuBA being replaced by dhfrA4 that confers resistance to
trimethoprim. A. xylosoxidans was detected in the environmental settings and

hospital environments suggesting that blajuB-\ is likely disseminated in the
Tripoli clinical setting and thus this work requires more studies and
surveillance to detect any further occurrence of 6 / « t m b - i and other MBL genes.
The occurrence of 6 / # t m b - i in the environment is worrisome as it has the
potential to colonise/infect patients and like other MBL genes; 6/ « v im , bla\Mp,
blawoM-i, blaom, blaspM-i, blasm-i, blaAm-i, ^#khm-i and ^/adim-i (Osano et
et al., 2010) has first appeared in non-fermenters.
The Km values for TMB-1 are similar to DIM-1 and GIM-1 for penicillins and
cephalosporins but are larger for meropenem indicating that meropenem is not
a “natural” substrate for TMB-1. Rather like many other MBLs, the origin of
TMB-1 will never be known. Any MBL should be regarded important even if
the kinetics are less impressive than previously reported MBLs, particularly
those encoded by mobile genes via ISCRs, transposons, ICEs or integrons
carried on plasmids. For example, 6 /# n d m -i has appeared in K. pneumoniae,
212
E. coli and other Enterobacteriaceae in the clinical setting as well as the
environment outside the hospitals and carried on a variety of different
plasmids with the immediate environment surrounding 6/andm-i highly
variable (Walsh et al., 2011; Poirel et al., 2011). It is the same scenario for the
dissemination of MBL encoding genes in environmental strains that share very
large genetic pool with numerous strains of bacteria possibly leading to the
emergence of MBLs in clinical strains originating from the environment.
The findings of this study show the presence of several antibiotic resistance
mechanisms among Enterobacteriaceae and non-fermenters in Libya as shown
by the incidence o f clonally and non-clonally related K. pneumoniae and E.
coli carrying blacix-u-\5^ E c p l , the dissemination of class 1 integrons that
carry MBL genes and other P-lactamase genes as well as aminoglycoside and
trimethoprim resistance genes. This study, moreover, tried to associate the
emergence of ESBLs and other resistance mechanisms with clonality to
further our understanding on how the transmission of antimicrobial resistance
in Libyan hospitals occurs. The study also determined the MBLs, VIM-2 and
TMB-1, as responsible for high-level p-lactam resistance in non-fermenters,
P. aeruginosa and A. xylosoxidans shown in the clinical and non-clinical
settings and health care facilities in Tripoli and Benghazi.
It is worth mentioning that acute care facilities are among the most common
sites for the development of antimicrobial resistance. The intensive uses of
213
antimicrobial agents as well as suboptimal infection control policies are major
factors affecting the emergence and transmission of antibiotic resistance
bacteria in Libyan hospitals. Antibiotic resistance genes can be used as a
genetic marker for bacterial outbreaks and help in the early detection of
outbreaks bacterial infections and thus might effectively reduce the cost of
managing outbreaks, decrease morbidity and mortality by eliminating the
nosocomial pathogens and consequently enhance the application of infection
control programs. In contrast Libya has few of these capabilities even before
the civil war and the data produced in this thesis are the first studies of this
kind to be undertaken. Prior to my research, there were no reports of resistance
genes, mobile elements. Libyan hospitals lack a systematic approach to patient
management - for example, hospitals in Benghazi can get
ampicillin/sulbactum but hospitals in Tripoli could not. Microbiology
laboratories were also desperately inadequate with no standardization of media
or susceptibility testing. Infection control programs were also absent with no
instructions to clinical staff on decreasing infections and, hand-washes and
disposal towels are completely absent.
Thus, it is hoped that the data from this thesis together with a new political
beginning for Libya can help build an awareness of resistance and how the
monitoring of resistance genes and mobile genetic elements can aid and
enhance patient outcome. The potential to improve the laboratory and clinical
infra-structure in Libya is enormous and, with an awareness of operating
214
systems in laboratories in other countries, together with a desire to be more
transparent in implementing them, patient outcome will hopefully be
dramatically improved.
I trust and hope the data from my thesis is merely the beginning.
215
Chapter Seven
General Discussion
This study investigated the mechanism of antibiotic resistance in randomly
collected isolates of Gram-negative bacteria in Tripoli and Benghazi (Figure
7.1). The isolates were from clinical samples recovered from patients admitted
to the hospitals and from the hospital environment and for the purpose of my
thesis these are regarded as non-clinical samples. These swabs were from
floors, walls, bedsides, toilets, workstation tables, mechanical ventilators,
oxygen suppliers, stainless steel containers, curtains, baby incubators, trolleys
and other instruments used in the hospitals in particular ICUs. The non
hospital environmental isolates were swabs collected from streets in Tripoli
and Benghazi; the samples were from floors and dusty areas in the streets.
parnah
Figure 7.1 Map of Libya f. -.Tpbruk
showing the important
cities and their locations eg ypt

Sabha
(http://www.google.co.uk/imgres?q=li
byan+map&h l=en&gbv=2&tbm=isch Al Jawf
NIGER
ISO 300 fem CHAD SUDAN

300 mi
The data in my thesis clearly demonstrates the emergence of MDR Gram-
negative bacteria in hospital settings that include clinical and non-clinical
isolates. In total, 171 isolates were recovered for this study.
205
Enterobacteriaceae represent the most numerous of the Gram-negative with K.
pneumoniae as the most frequently isolated bacteria. High prevalence of
ESBLs was detected among K. pneumoniae and E. coli, more importantly
blacix-u group 1 were found widely disseminated in association with ISEcpl
being located immediately upstream o f most of the ESBL encoding gene.
The highest incidence o f blact x - m group 1 was detected among clinical and
non-clinical isolates collected from hospitals. K. pneumoniae and E. coli
isolates tested for the occurrence o f conjugative plasmids responsible for the
movement o f blacjx-u group 1/ISE cpl. The study showed that these plasmids
can move blacix-u-\5^ E c p l genes from the resistant isolates to sensitive E.
coli, leading to E. coli transconjugants expressing an MDR phenotype. It is
worth mentioning that plasmid mediated 6 /« c tx -m -i5 associated with ISEcpl
has been detected in an environmental isolate of K. pneumoniae AES817
(ST511) cultured from one o f Benghazi streets - this isolate clearly has not
been recently exposed to antibiotics. These findings are an alarmingly
indication that an outbreak of MDR K. pneumoniae and E. coli isolates can
occur in different hospitals that provide different services to patients; these
hospitals are maternity hospitals, pediatric hospitals, surgical hospitals and
general hospital in Tripoli and Benghazi.
The occurrence o f K. pneumoniae and E. coli in Libyan hospitals is interesting
in terms o f the prevalence o f clonally and non-clonally related blacix-u -\5
206
positive isolates (Lee et al., 2011; Webster et al., 2011; Alfaresi et al., 2011;
Fam et al., 2011; Al Sweih et al., 2011). The collection of isolates being
clinical, non-clinical or environmental was non-representative seeking the
occurrence o f any resistance mechanism in these isolates. Among all the
isolates positive for blacix-u group 1, 4 pairs o f K. pneumoniae isolates were
found clonally related. One pair represented two isolates that were found in
two different hospitals in Benghazi - isolates AES 135 and AES 140 that were
cultured from urine and blood samples, respectively.
The occurrence o f several clonally related K. pneumoniae and E. coli that
resulted from the non-representative sample collection reflected the total lack
of appropriate infection control programs in Libyan hospitals. Lack of hospital
hygiene is another reason that has contributed to the emergence and spread of
multi-drug resistant isolates o f K. pneumoniae and E. coli in Libyan hospitals.
The overuse o f extended- spectrum cephalosporins to treat infections in Libya
may be another significant factor for the appearance o f clonally and non-
clonally related isolates o f K. pneumoniae and E. coli in Libya. (Ito &
Kamimura, 2011; Wang et al., 2011)
The occurrence of blacix-u group 1USEcp 1on different plasmid sizes in K.
pneumoniae and E. coli isolates suggest the movement of these genes by
conjugative plasmids (Lavollay, et al., 2006) as shown from data on mating
studies in a subset of these isolates. The ability o f these isolates to mobilize
207
and facilitate the spread of antibiotic resistance genes may explain the
frequency of blacTx-M groupl/ISiscpi in non-clonally related species o f K.
pneumoniae and E. coli (Woodford et al., 2004; Gonullu et al., 2008;
Lavollay et al., 2006, Yu & K. Cheng, 2004; Ramdani-Bouguessa, et al.,
2006; Abbassi et al., 2008). A possible explanation for the different plasmid
location o f blacix-u group 1 is due to the presence of the insertion sequence,
ISEcpl that is known to move and promote the expression o f the ESBL gene.
Two suggested mechanisms o f blacix-u group 1 acquisition are proposed: the
movement of blacix-u group 1 by conjugative plasmids and the role o f the
insertion sequence ISEcpl in mobilizing blacix-u group 1 within the strains
(Abbassi et al., 2008; Poirel et al., 2003; Naseer & Sundsfjord, 2011; Younes
et al., 2011; Gonullu et al., 2008; Villa et al., 2010; Partridge et al., 2011).
Data on Libyan E. coli give evidence o f the occurrence o f both mechanisms in
the movement o f antibiotic resistance determinants for two reasons, blacix-u
group 1 and ISEcpl were detected on two different plasmid locations in parent
and recipients which suggests the role o f ISEcpl in mobilizing blacix-u
groupl (Rejiba et al., 2011; Smet et al., 2010) to a different size plasmid in
recipient. An alternative suggestion is the creation of a co-integrative plasmid
during conjugation. Similar resistance profiles were detected in parents o f K.
pneumoniae and the E. coli transconjugants (GFP E. coli and E. coli J53)
suggesting that many o f the antibiotic resistance determinants expressed by
208
the Libyan isolates o f K. pneumoniae and E. coli are located on conjugative
plasmids (M nif et al., 2010; Cullik et al., 2010; Partridge et al., 2011).
In addition to previously reported K. pneumoniae clones known to carry
6/<2c t x -m - i 5 e.g. ST15, ST29, ST 101 and ST 147, this study provided new
MLST groups - K. pneumoniae ST509 and ST486 and, additionally, a novel
environmental allele ST511 was found carrying blacix-u -\5 on different
plasmid sizes. The results o f MLST data provided further evidence of the
incidence o f 6/<2c tx -m - is in different clones of K. pneumoniae and also shows
novel sequence types are involved in the carriage o f 6/<2ctx -m - i 5 in Libya
(Nielsen et al., 2011; Pitart et al., 2011; Papagiannitsis et al., 2011; Hrabak et
al., 2009; Damjanova et al., 2008).
RAPD and MLST techniques used in this study show that they are similar in
detecting the strain relatedness between K. pneumoniae isolates; however,
they still are less discriminatory than PFGE which is based on genomic DNA
separation rather than amplification o f specific fragments or housekeeping
genes. This study supports the application of RAPD technique to correlate the
relation o f bacterial species to each other but not in determining the detailed
clonality within a species. Unsurprisingly, RAPD and MLST when compared
with PFGE demonstrated quite different results - some isolates appeared very
similar by RAPD but distinctly different with PFGE. Other isolates e.g. K.
pneumoniae AES74, AES59 and AES 1029 that belong to ST15 were
209
comparable by RAPD and MLST, but by PFGE demonstrated a low-level of
similarity. However, RAPD may help in the general assessment to determine
the broad similarity among bacterial species and it is rapid and inexpensive.
The MLST method is reliable but depends upon sequence stability among
housekeeping genes. PFGE can provide very detailed information on the
differences between species or subspecies; but it is specialized, temperamental
and quite expensive compared to MLST (Hotchkiss et al., 2011).
Class 1 integrons, whether alone or in association with Tn402-type or Tn27
transposons are among the most represented mobile genetic elements
responsible for capturing genes in the form of gene cassettes. Among the
Libyan isolates tested, 11 different type o f class 1 integrons were detected in
21 isolates o f Enterobacteriaceae and non-fermenters. Twelve class 1
integrons were detected in K. pneumoniae, 3 in E. coli, 3 in P. aeruginosa and
2 in A. xylosoxidans. Most integrons detected in this study carried
trimethoprim and aminoglycoside resistance genes which are typically found
as gene cassettes. Six different integrons were found in K. pneumoniae
isolates, two of which were embedded on Tn402-type transposons, o f these 6
integrons, 5 integrons have been previously reported from different
geographical areas. One of these integrons {Inti, dfrA17, aadA5, qacEA) was
also found in K. pneumoniae and E. coli isolates examined in this study
(Vinue et al., 2008; Tang et al., 2011). Another integron harbouring dfrA\2
and aadA2 was detected in K. pneumoniae clinical isolate AES48. This isolate
210
was identified in this study as ST 147 which is known as a world wide ST
contributing to the emergence of ^/actx-m-is genes. The genetic context of this
integron has been reported in clinical isolates o f E. coli in Asia and Europe
(Yu et al., 2004; Tang et al., 2001; Saenz et al., 2009; Vinue et al., 2008)
These findings show that the same resistance mechanisms are disseminated
worldwide, and also show that the Libyan isolates share the same genetic pool
with bacterial species worldwide.
Class 1 integrons detected in non-fermenters included in addition to
trimethoprim and aminoglycoside resistance genes, antibiotic resistance genes
responsible for conferring resistance to broad-spectrum (3-lactams. blay\u-i
was detected in two clonal isolates of P. aeruginosa collected from different
places in the same hospital, AES 81 was non-clinical sample found in a
stainless steel container and AES83 was recovered from a clinical sample
cultured from a tip of a catheter. These findings show the potential of P.
aeruginosa to acquire class 1 integrons in Libya and that it is widespread both
in clinical and environmental isolates as have been reported worldwide
(Valenza et al., 2010; Rojo-Bezares et al., 2011; Guevara, A. et al., 2009; Van
der Bij et al., 2011; Piyakul et al., 2011).
Perhaps the most surprising finding from this study was ^/«tmb-i, a novel
MBL gene discovered in A. xylosoxidans cultured from Tripoli central
211
hospital. 6/atmb-i has been detected as a gene cassette embedded in the first
position of class 1 integron followed by an aminoglycoside resistance gene
(aacA4) and oxacillinases gene (blaoxAA)- The same A. xylosoxidans isolate
possessed a second near-identical integron composed of aacA4, blaoxAA but
with blajuB-\ being replaced by dhfrA4 that confers resistance to
trimethoprim. A. xylosoxidans was detected in the environmental settings and
hospital environments suggesting that blajuB-\ is likely disseminated in the
Tripoli clinical setting and thus this work requires more studies and
surveillance to detect any further occurrence of blajuB-i and other MBL genes.
The occurrence of blaj m b - i in the environment is worrisome as it has the
potential to colonise/infect patients and like other MBL genes; bla^m, bla\u?,
blamm-u blaQiu, &/«spm-i, blasm-u blaaim-i, ^/«khm-i and bladim-i (Osano et
et al., 2010) has first appeared in non-fermenters.
The Km values for TMB-1 are similar to DIM-1 and GIM-1 for penicillins and
cephalosporins but are larger for meropenem indicating that meropenem is not
a “natural” substrate for TMB-1. Rather like many other MBLs, the origin of
TMB-1 will never be known. Any MBL should be regarded important even if
the kinetics are less impressive than previously reported MBLs, particularly
those encoded by mobile genes via ISCRs, transposons, ICEs or integrons
carried on plasmids. For example, 6/andm-i has appeared in K. pneumoniae,
212
E. coli and other Enterobacteriaceae in the clinical setting as well as the
environment outside the hospitals and carried on a variety of different
plasmids with the immediate environment surrounding 6/<?n d m - i highly
variable (Walsh et al., 2011; Poirel et al., 2011). It is the same scenario for the
dissemination o f MBL encoding genes in environmental strains that share very
large genetic pool with numerous strains of bacteria possibly leading to the
emergence o f MBLs in clinical strains originating from the environment.
The findings of this study show the presence of several antibiotic resistance
mechanisms among Enterobacteriaceae and non-fermenters in Libya as shown
by the incidence o f clonally and non-clonally related K. pneumoniae and E.
coli carrying 6 /<2c t x - m - i 5/lSEcp 1 , the dissemination of class 1 integrons that
carry MBL genes and other p-lactamase genes as well as aminoglycoside and
trimethoprim resistance genes. This study, moreover, tried to associate the
emergence o f ESBLs and other resistance mechanisms with clonality to
further our understanding on how the transmission of antimicrobial resistance
in Libyan hospitals occurs. The study also determined the MBLs, VIM-2 and
TMB-1, as responsible for high-level p-lactam resistance in non-fermenters,
P. aeruginosa and A. xylosoxidans shown in the clinical and non-clinical
settings and health care facilities in Tripoli and Benghazi.
It is worth mentioning that acute care facilities are among the most common
sites for the development of antimicrobial resistance. The intensive uses of
213
antimicrobial agents as well as suboptimal infection control policies are major
factors affecting the emergence and transmission of antibiotic resistance
bacteria in Libyan hospitals. Antibiotic resistance genes can be used as a
genetic marker for bacterial outbreaks and help in the early detection of
outbreaks bacterial infections and thus might effectively reduce the cost of
managing outbreaks, decrease morbidity and mortality by eliminating the
nosocomial pathogens and consequently enhance the application of infection
control programs. In contrast Libya has few o f these capabilities even before
the civil war and the data produced in this thesis are the first studies of this
kind to be undertaken. Prior to my research, there were no reports of resistance
genes, mobile elements. Libyan hospitals lack a systematic approach to patient
management - for example, hospitals in Benghazi can get
ampicillin/sulbactum but hospitals in Tripoli could not. Microbiology
laboratories were also desperately inadequate with no standardization of media
or susceptibility testing. Infection control programs were also absent with no
instructions to clinical staff on decreasing infections and, hand-washes and
disposal towels are completely absent.
Thus, it is hoped that the data from this thesis together with a new political
beginning for Libya can help build an awareness of resistance and how the
monitoring o f resistance genes and mobile genetic elements can aid and
enhance patient outcome. The potential to improve the laboratory and clinical
infra-structure in Libya is enormous and, with an awareness of operating
214
systems in laboratories in other countries, together with a desire to be more
transparent in implementing them, patient outcome will hopefully be
dramatically improved.
I trust and hope the data from my thesis is merely the beginning.
215
Chapter Eight
Appendices
Appendix A
Table A .l Antibiotics and chemicals used in selection of antibiotic
resistant strains experiments
Antibiotic Source
Ceftazidime SIGMA-ALDRICH
Rifampicin SIGMA-ALDRICH
Meropenem SIGMA-ALDRICH
Ertapenem SIGMA-ALDRICH
Imipenem SIGMA-ALDRICH
Cefoxitin SIGMA-ALDRICH
Cefuroxone SIGMA-ALDRICH
Ampicillin SIGMA-ALDRICH
Piperacillin SIGMA-ALDRICH
Sodium azide SIGMA-ALDRICH
Kanamycin SIGMA-ALDRICH
217
Appendix A
Table A.2 Oligonucleotides used for PCR amplification and DNA

sequencing
Gene target Primer name Primer sequence Reference

6/nrCTX-M15 CTX-M-15 F 5'GTTCACGCTGATGGCGACGGC'3 This study
6/oCTX-M-15 CTX-M-15 R 5'GACGCTAATACATCGCGACGGC'3 This study
Beginning of Leflon-Guibout
ISEcpu2 5' AATACTACCTTGCTTTCTGA '3
ISEcpl et al., 2004
The end o f Ho et al., 2005
ISEcpul 5' AAA A ATG ATTG A AAGGTGGT'3
ISEcpl
Levesque et al.,
Integrase gene VAF 5 'GCCTGTTCGGTTCGTAAGCT'3
1994
Transposase Toleman et al.,
tniC 5 'CG ATCTCTGCG AAG AACTCG'3
gene 2007
Quaternary Levesque, C. et
Ammonium QacR 5'CGGATGTTGCGATTACTTCG'3 al., 1994
Compound
Mammeri, H. et
Transposon TniC 5 'CG ATCTCTGCG A AG AACTCG'3
al., 2003
Kiratisin et al.,
blaTEM TEM F 5 'TCCGCTC ATG AG AC A ATAACC'3
2008
Kiratisin et al.,
blaTEM TEM R 5'TTGGTCTGACAGTTACCAATGC'3
2008
Kiratisin et al.,
blaSH V SHV F 5'TGGTTATGCGTTATATTCGCC'3
2008
Kiratisin et al.,
blaSH V SHV R 5'GGTTAGCGTTGCCAGTGCT'3
2008
Lee, K. et al.,
ISCR\ ISCRl F 5'GGT TGC AAC GAC TCA AGCG'3
2005
Lee, K. et al.,
ISCRl ISCRl R 5'CAC TCG TTT ACC GCT CAA GC'3
2005
5'GCT GCT CAA GGA GCA CAG Brolund et al.,
blaam pC MOX MOX F 2010
GAT'3
5'CAC ATT GAC ATA GGT GTG GTG Brolund et al.,
blaam pC MOX MOX R 2010
C'3
5'TGG CCA GAA CTG AC A GGC AAA Brolund et al.,
blaam pC CITM CITM F 2010
'3
Brolund et al.,
blaam pC CITM CITM R 5'TTT CTC CTG AAC GTG GCT GGC'3
2010
blaam pC 5'AAC TTT CAC AGG TGT GCT Brolund et al.,
DHAM F 2010
DHAM GGGT'3
blaam pC Brolund et al.,
DHAM R 5'CCG TAC GCA TAC TGG CTT TGC'3
DHAM 2010
blaam pC 5'AAC AGC CTC AGC AGC CGG Brolund et al.,
ACCM F 2010
ACCM TTA'3
blaam pC Brolund et al.,
ACCM R 5'TTC GCC GCA ATC ATC CCT AGC'3 2010
ACCM
5'TCG GTA AAG CCG ATG TTG CGG Brolund et al.,
blaam pC EBCM EBCM F 2010
'3
Brolund et al.,
blaam pC EBCM EBCM R 5 'CTT CCA CTG CGG CTG CCA GTT'3
2010
5'AAC ATG GGG TAT CAG GGA GAT Brolund et al.,
blaam pC FOXM FOXM F 2010
G'3
5'CAA AGC GCG TAA CCG GAT Brolund et al.,
blaam pC FOXM FOXM R 2010
TGG'3
218
Table A.3. Oligonucleotides used for PCR amplification and DNA
sequencing
G ene target P rim er nam e P rim er sequence Reference

TMB-1 T rip-1F61 5'G C C A AC G A A G AA AT A CCC G C ’3 This study
TMB-1 T rip2-10 5'T G G G C T A G G T TA CAC TGG TG'3 This study
TMB-1 T rip617R 5'T T C TA G C G G A TT GTG G CC AC'3 This study
TMB-1 T rip2 5'C A A G GA G C T C A T TCA AAGG'3 This study
TMB-1 T rip l 5 'G G A G CA GGC A AG GAG CT’3 This study
TMB-1 T rip4 5 'A A G G G T T A A C A A GTG G CA GC'3 This study
TMB-1 T rip75 5 'A CC C G G A T T G GA A G T TGA GG'3 This study
TM B-1 T rip l FF 5'T G A T C A G TG GCC A CA A TC C G ’3 This study
TM B-1 T rip l F 5'C G G A T T G TG GCC A CT G AT C A ’3 This study
TM B-1 Trip3 5'G G C C A T A C T A A T G AT A AC’3 This study
dhfrA dhffA 1FR 5 'C C C G A T A A C TCC A TT C TT C G ’3 This study
dhfrA dhfrA IF 5'C G A A G A A T G G AG TTA TCG GG'3 This study
dhfrA dhfrA 1R 5'G T T A G A G GC G A A GTC TTG G G ’3 This study
dhfrA dhfrA IF F 5 'C C C A A G A C T TCG C C T CTA A C ’3 This study
aac6II aac6II R 5'G G C G TC G G C TTG AAT GAG TT ’3 This study
aac6II aac6II F 5'A A G T G G C A G C A A CGG A TT C G ’3 This study
aac611 aac6II FR 5 'G A A TC C G T T G CT GCC A C T T G ’3 This study
aac61I aac6II FF 5'C A A C TC A T T C A A GCC GAC G C’3 This study
aac6II aac6II FR 5'G T G C TC G CG GAC ATG A AA T G ’3 This study
O xa-4 O xa-4-F R 5'C A C T T A TG G C A T TTG A TG C G ’3 This study
Oxa-4 O xa-4-F 5'C G C A TC A A A TGC C A T A AG T G ’3 This study
S P M -1 D F 6/tfSPM -l 5'C C T A C A A TC TA A CGG CGA CC ’3 Zavascki et al., 2005
SD PM -1D W aSPM -1 5'T C G C C G T G T C CA G GT A TA A C ’3 Zavascki et al., 2005
R
IM P-2 F b la \ U ? - 2 5'G G C A G T C G C C C T A AA A CA A A ’3 W u et al., 2007
IM P2 R bla \M P -2 5 'TA G T TA C TT GGC TG T G A T G G ’3 Wu et al., 2007
IMP-1 R 6/a IM P -l 5'T T A G TT G C T TGG TTT TG A TG ’3 Queenan & Bush, 2007
IMP-1 F b la \M P -\ 5'T G A G C A A G T T A T CTG T A T TC ’3 Queenan & Bush, 2007
V IM -F b la V IM 5'G T C T A T TTG A CC GCG TC ’3 Cezario et al., 2009
V IM - R 6/aV IM 5'C T A C TC A A C G AC TGA G CG ’3 Cezario et al., 2009
G IM - F 6/a G IM -l 5'A G A A C C T TG A CC GAA C G C A G ’3 Queenan & Bush, 2007
G IM -R b la G \M -\ 5'A C T C A T G A C T C C T C A C G A G G ’3 Queenan & Bush, 2007
NDM F 6/aN D M -l 5 'G A A G C T G A G CAC CGC A TTA G ’3 Sidjabat et al., 2010
NDM R 6/a N D M -l 5'T G C G G G C CG TA T GAG TG A T T ’3 Sidjabat et al., 2010
DIM-1 F 6/a D IM -l 5' G CT T G T C TT CGC TTG C TA ACG Poirel et al., 2011
’3
DIM-1 R bIaD lM -\ 5' C G T TCG G C T GGA TTG A TT TG ’3 Poirel et al., 2011
SIM-1 F 6/a S IM -l 5' TAC AAG G G A T T C G G C A TC G ’3 Poirel et al., 2011
SIM-1 R 6/o S IM -l 5' TAA T G G C C T G T T C C C A TG T G Poirel et al., 2011
’3
KHM-1 F 6/aK F IM -l 5' G GT A TG C G C TG A C G A TTC ’3 Sekiguchi et al., 2008
KHM-1 R Zj/aKHM -l 5' T TT A T T TG G TGG C TG T TT T G T C Sekiguchi et al., 2008
’3
AIM-1 F bla A \M - \ 5' CTG A A G G TG TAC G GA A AC AC Poirel et al., 2011
’3
AIM-1 R b ia S lM -\ 5' G TT C G G C C A C C T C G A A TT G ’3 Poirel et al., 2011
219
Table A.4. Primers, target site and size of replicons tested
Plasm id D N A sequence T arget site Gene size (bp)

M ultiplex 1
HI1 FW 5 '-g g a gcg atg gat tac ttc agt ac 3
parA -parB 471
HI1 RV 5 '-tg c cgt ttc acc teg tg a g ta 3
HI2 FW 5 '-tttc tcc tg a gtc acc tg t taa cac 3
Iterons 644
HI2 RV 5 '-g g c tcac tac cgt tgt cat cct 3
11 FW 5'-cg a aag ccg gac ggc agaa 3
RNA1 139
11 RV 5 '-tcg tegt tcc gcc aag ttc g t 3
M ultiplex 2
X FW 5 '-aac ctt ag a ggc tat tta agt tg c tgat
'3
ori y 376
X RV 5 '-tg a gag tea att ttt ate tea tgt ttt age
'3
L/M FW 5 '-g g a tg a aaa e ta tea g ca tct g aa g '3
repA ,B ,C 785
L/M RV 5 '-ctg cag g g g cga ttc ttt agg '3
N FW 5V -gtc taa cg a get tac cga ag '3
repA 559
N RV 5 '-g tt tea act ctg cca agt tc '3
M ultiplex 3
F1A FW 5 '-cca tget ggt tct aga gaa g gtg '3
Iterons 462
F IA R V 5 '-g ta tat cct tac tgg ctt ccg cag '3
FIB FW 5 '-g g a gtt ctg aca cac gat ttt ctg '3
repA 702
FIB RV 5 '-ctc ccg teg ctt cag ggc att '3
W FW 5 '-cct aag aac aac aaa gcc cccg '3
repA 242
W RV 5 '-g g t gcg eg g cat ag a acc gt '3
M ultiplex 4
Y FW 5 '-a a t te a aac aac act g tg cag cctg '3
Y RV 5 '-g cg aga atg gac gat tac aaa act tt repA 765
'3
P FW 5 '-eta tgg ccc tgc aaa ege gcc ag a aa
'3 Iterons 534
P RV 5 '-tca ege gcc agg g cg cag cc '3
F IC F W 5 '-g tg aac tgg cag atg agg aagg '3
repA2 262
FIC RV 5 '-ttc tcc teg teg cca aac tag at '3
M ultiplex 5
A/C FW 5 '-g ag aac caa ag a caa ag a cct gga
3' repA 465
A/C RV 5 '-acg aca aac ctg aat tgc etc ctt '3
T FW 5 '-ttg gcc tgt ttg tgc eta aac cat '3
repA 750
T RV 5 '-cg t tga tta cac tta get ttg gac '3
F IIsF W 5 '-ctg teg ta a get gat ggc '3
repA 270
FIIs RV 5 '-ctc tgc cac aaa ctt cagc'3
Sim plex 1
Frep FW 5 '-tg a teg ttt aag gaa ttt tg '3
R N A l/rep A 270
Frep RV 5 '-g aa gat cag tea cac cat cc '3 .
Sim plexs 2 and 3

K/B FW 5 '-g cg gtc egg aaa gcc ag a aaac '3
RNA1 160
K/B RV 5 '-tct ttc acg age ccg cca aa '3
B/O RV 5 '-tct gcg ttc ege caa gtt cga '3 RNA1 159
220
Appendix B
Figures of multiplex PCR to detect the occurrence of CTX-M type ESBLs

in K. pneumoniae (Figures B.1-B.5 in the next three pages)
bp
bp
1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 18 19 20
Figure B.l Multiplex PCR experiment to detect the incidence of CTX-M

type ESBLs groups 1,2,8,9 and 26 in K. pneumoniae isolates. Lanel: Marker.
Lane2: AES152. Lane3: AES170. Lane4: AES172. Lane5: AES178. Lane6:
AES 117. Lane7: AES197. Lane8: AES187. Lane9: AES188. LanelO:
AES 194. Lanel 1: AES 203. Lanel2: AES216. Lanel3: AES225. Lanel4:
AES236. Lanel5: AES258. Lanel6: AES260. Lanel7: AES261. Lanel8:
AES265. Lanel9: AES268. Lane20: AES270.
400 bp
200 b p
Figure B.2 Multiplex PCR experiment to detect the incidence of CTX-M type
ESBLs groups 1,2,8,9 and 26 in K. pneumoniae isolates. Lanel: Marker.
Lane2: AES271. Lane3: AE273. Lane4: AES274. Lane5: AES275. Lane6:
AES279. Lane7: AES280. Lane8: AES917. Lane9: AES942. LanelO: H20.
Lane 11: Marker.
221
400 bp
200 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Figure B.3 Multiplex PCR experiment to detect the incidence of CTX-M

type ESBLs groups 1,2,8,9 and 26 in K. pneumoniae isolates. Lanel:
Marker. Lane2: AES506. Lane3: AES722. Lane4: AES808. Lane5:
AES939. LanelO: AES943. Lanel 1: AES960. Lanel2: AES961. Lanel3:
AES970. Lane 14: AES973. Lanel5: AES975. Lanel6: AES977.
'•<C.K • '
400 bp
200 bp
1 2 3 4 5 6 7 8 9 1ft 11 12 13 14 15 16 17 18 19
Figure B.4 Multiplex PCR experiment to detect the incidence of CTX-

M type ESBLs groups 1,2,8,9 and 26 in K. pneumoniae isolates. Lanel:
Marker. Lane2: AES982. Lane3: AES983. Lane4: AES984. Lane5:
AES985. Lane6: AES987. Lane7: AES994. Lane8: AES 1001. Lane9:
AES 1004. LanelO: AES 1004(1). Lanel 1: AES1013. Lanel2:
AES1025. Lanel3: AES1026. Lanel4: AES1028. Lanel5: AES1029.
Lanel6: AES1036. Lanel7: AES1052. Lanel8: AES1053. Lanel9.
Lane 19: Marker.
222
400 bp
200 b p
1 2 3
Figure B.5 Multiplex PCR experiment to detect the incidence of CTX-M type
ESBLs groups 1,2,8,9 and 26 in K. pneumoniae isolates. Lanel: Marker.
Lane2: Positive control AES 140). Lane3: Negative control (H20). Lane4:
Marker
2 23
PFGE of 51 digestion of some isolates of K. pneumoniae and probing with
blact x -m -15 (Figures B.6 and B.7, next two pages)
§ »*» m* %
350 kb
300 kb
250 kb
200 kb
150 kb
100 kb
50 kb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure B.6 PFGE o f 51 digests for K. pneumoniae AES isolates. Lanel:

Marker. Lane2: AES809. Lane3: AES817. Lane4: AES203. Lane5: AES836.
Lane6: AES939. Lane7: AES961. Lane8: AES942. Lane9: AES 188. LanelO:
AES994. Lanel 1: AES960. Lanel2: AES970. Lanel3: AES975. Lanel4:
AES977. Lanel5: AES982.
224
400 kb
350 kb
300 kb
250 kbj
200 kb
150 kb
100 kb
1 2 3 4 5 6 7 8 9 10 II 12 13 14 15
Figure B.7 Autorad after probing with blacix-uASÊcpl of blotted PFGE

from fig B6. Lanel: Marker. Lane2: AES809. Lane3: AES817. Lane4:
AES203. Lane5: AES836. Lane6: AES939. Lane7: AES961. Lane8: AES942.
Lane9: AES188. LanelO: AES994. Lanel 1: AES960. Lanel2: AES970.
Lanel3: AES975. Lanel4: AES977. Lanel5: AES982.
225
DNA sequences from class 1 integrons of some of K. pneumoniae
Figure legend is above the figure.
Figure B.8 DNA sequence of K. pneumoniae AES59 amplified by VAF

primer.
AACCTTGACCGAACGCAGCGGTGGTAACGGCGCAG
TGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCAT
CCA
AGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGA
TGTTA
CGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATTAAGGGAGTTAAATTGA
AAATAT
CATTGATTTCTGCAGTGTCAGAAAATGGCGTAATCGGTAGTGGTCCTGATATCCC
GTGGT
CAGTAAAAGGTGAGCAACTACTCTTTAAAGCGCTCACATATAATCAATGGCTCCT
TGTCG
GAAGAAAAACATTTGACTCTATGGGTGTTCTTCCAAATCGCAAATATGCAGTAGT
GTCAA
AGAACGGAATTTCAAGCTCAAATGAAA
Figure B.9 Alignment o f DNA from figure B.8 with DNA from gene bank
Matched w ithDfrA17
>>EM_PRO: F J 8 9 5 3 0 1 F J 8 9 5 3 0 1 . 1 S h i g e l l a f l e x n e r i p l a s m i d
unknow n
c l o n e 0 5 1 0 0 c l a s s 1 i n t e g r o n DNA i n t e g r a s e i n t l l ( i n t l l ) ,
d i h y d r o f o l a t e r e d u c t a s e D fr A 1 7 ( d f r A 1 7 ) , a n d
a m i n o g l y c o s i d e - 3 1- a d e n y l y l t r a n s f e r a s e (a a d A 5 ) g e n e s ,
c o m p le t e c d s . (2 8 1 3 n t)
i n i t n : 2 1 1 0 i n i t l : 2 1 1 0 o p t : 2 1 1 0 Z - s c o r e : 2 1 7 8 .0 b i t s :
4 1 4 .8 E (1 4 2 4 3 9 2 4 6 ): 7 .8 e - 1 1 2
b a n d e d S m it h - W a t e r m a n s c o r e : 2 1 1 0 ; 10 0 .0 % i d e n t i t y (100.0%
s im ila r ) in 422 n t o v e r la p (1 -4 2 2 :1 0 6 3 -1 4 8 4 )
10 2 0 3 0
EMBOSS AACCTTGACCGAACGCAGCGGTGGTAACGG
EM_PRO
GTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAAC
GG
1040 1050 1060 1070 1080 1090
40 50 60 7 0 80 90
EMBOSS
CGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGG
GC
EM PRO
226
CGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGG
GC
1100 1110 1120 1130 1140 1150
100 110 120 130 140 150

EMBOSS
ATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAAC
GA
EM_PRO
ATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAAC
GA
1160 1170 1180 1190 1200 1210
160 170 180 190 2 00 210

EMBOSS
TGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATTAAGGGAGTTAAATTG
AA
EM_PRO
TGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATTAAGGGAGTTAAATTG
AA
1220 1230 1240 1250 1260 1270
220 230 240 250 260 270

EMBOSS
AATATCATTGATTTCTGCAGTGTCAGAAAATGGCGTAATCGGTAGTGGTCCTGATATC
CC
EM_PRO
AATATCATTGATTTCTGCAGTGTCAGAAAATGGCGTAATCGGTAGTGGTCCTGATATC
CC
1280 1290 1300 1310 1320 1330
280 290 300 310 320 330

EMBOSS
GTGGTCAGTAAAAGGTGAGCAACTACTCTTTAAAGCGCTCACATATAATCAATGGCTC
CT
EM_PRO
GTGGTCAGTAAAAGGTGAGCAACTACTCTTTAAAGCGCTCACATATAATCAATGGCTC
CT
1340 1350 1360 1370 1380 1390
340 350 360 370 380 390

EMBOSS
TGTCGGAAGAAAAACATTTGACTCTATGGGTGTTCTTCCAAATCGCAAATATGCAGTA
GT
EM_PRO
TGTCGGAAGAAAAACATTTGACTCTATGGGTGTTCTTCCAAATCGCAAATATGCAGTA
GT
1400 1410 1420 1430 1440 1450
400 410 420

EMBOSS GTCAAAGAACGGAATTTCAAGCTCAAATGAAA
227
EM_PRO
GTCAAAGAACGGAATTTCAAGCTCAAATGAAAACGTCCTAGTTTTTCCTTCAATAGAA
AA
1460 1470 1480 1490 1500 1510
EM_PRO
TGCTTTGAAAGAGCTATCAAAAGTTACAGATCATGTATATGTCTCTGGCGGGGGTCAA
AT
1520 1530 1540 1550 1560 1570
f7 EM PRO FN568 Kluyvera georgiana 1087 211 100. 100. 1

2 351 conjugative IncFII plasmid 2 0 0 0
pTC10 (partial) harboring a
c la ss 1 integron (dfrA17 and
aadA5 g e n e ca ssettes), Tn3-
bla(TEM-1b)-IS26 and Tn21
(partial)
Cross-references and related
information in:
• Nucleotide S eq u en ce
s
• Protein Families
• O ntologies
• Protein S e q u e n c e s
>>EM_PRO: F N 5683 5 1 FN5 6 83 5 1 . 1 K lu y v e r a g e o r g i a n a
c o n ju g a tiv e
I n c F I I p l a s m i d pTCIO ( p a r t i a l ) h a r b o r i n g a c l a s s 1
in te g r o n
(d fr A 1 7 an d aadA 5 g e n e c a s s e t t e s ) , T n 3 -b la (T E M -lb )-
IS 2 6 and
T n 21 ( p a r t i a l ) (1 0 8 7 2 n t)
in it n : 2110 i n i t l : 2110 o p t: 2110 Z -sc o r e : 2165.2
b i t s : 4 1 4 .3 E (142439246) : l e - 1 1 1
b a n d e d S m it h - W a t e r m a n s c o r e : 2 1 1 0 ; 1 0 0. 0 % i d e n t i t y
(100.0% s i m i l a r ) i n 422 n t o v e r la p ( 1 - 4 2 2 : 6 6 0 2 - 7 0 2 3 )
10 2 0 30
EMBOSS AACCTTGACCGAACGCAGCGGTGGTAACGG
EM_PRO
GTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGG
TAACGG
6580 6590 6600 6610 6620 6630
4 0 50 60 70 8 0 90
EMBOSS
CGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCC
TCGGGC
EM_PRO
CGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCC
TCGGGC
6640 6650 6660 6670 6680 6690
100 110 120 130 140 150

EMBOSS
ATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAG
CAACGA
228
EM__PRO
ATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAG
CAACGA
6700 6710 6720 6730 6740 6750
160 170 180 190 200 210

EMBOSS
TGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATTAAGGGAGTTAA
ATTGAA
EM_PRO
TGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATTAAGGGAGTTAA
ATTGAA
6760 6770 6780 6790 6800 6810
220 230 240 250 260 270

EMBOSS
AATATCATTGATTTCTGCAGTGTCAGAAAATGGCGTAATCGGTAGTGGTCCTGA
TATCCC
EM_PRO
AATATCATTGATTTCTGCAGTGTCAGAAAATGGCGTAATCGGTAGTGGTCCTGA
TATCCC
6820 6830 6840 6850 6860 6870
280 2 90 3 00 310 320 330

EMBOSS
GTGGTCAGTAAAAGGTGAGCAACTACTCTTTAAAGCGCTCACATATAATCAATG
GCTCCT
EM_PRO
GTGGTCAGTAAAAGGTGAGCAACTACTCTTTAAAGCGCTCACATATAATCAATG
GCTCCT
6880 6890 6900 6910 6920 6930
340 350 360 370 380 390

EMBOSS
TGTCGGAAGAAAAACATTTGACTCTATGGGTGTTCTTCCAAATCGCAAATATGC
AGTAGT
EM_PRO
TGTCGGAAGAAAAACATTTGACTCTATGGGTGTTCTTCCAAATCGCAAATATGC
AGTAGT
6940 6950 6960 6970 6980 6990
400 410 420

EMBOSS GTCAAAGAACGGAATTTCAAGCTCAAATGAAA
EM_PRO
GTCAAAGAACGGAATTTCAAGCTCAAATGAAAACGTCCTAGTTTTTCCTTCAAT
AGAAAA
7000 7010 7020 7030 7040 7050
EM_PRO
TGCTTTGAAAGAGCTATCAAAAGTTACAGATCATGTATATGTCTCTGGCGGGGG
229
TCAAAT
7060 7070 7080 7090 7100 7110
Figure B. 10 DNA sequence of K. pneumoniae AES59 1.5 kb amplified by

QacR primer
AGCCNGCCTTTCTGATATATCTCCCAATTTGTGTAGGGCTTATTATG
CACGCTTAAAAATAATAAAAGCAGACTTGACCTGATAGTTTGGCTGTGAGCAATT
ATGTG
CTTAGTGCA TCTA ACGCA TA GTTGA G CG GCG GG CGCAG CCCG TCCGCTTG A ACGC
CGAGT
TAGGCATCAGATGCCCTCGGCGCGGGTCGATGCACTTTTCGCACATGCCGCTCAA
CGCAA
GATTCTCTCAATCGTTGCTTTGGCATATCGAACGAACGCGGCCGTCTCTTCGACGC
GCAT
TGCTAGGTCGTCGTCCTCGCTACCCAGGTACGCCGCGCGTGCCTTGCAGATGAGG
GGCCG
ATGCTCGGCAGGCAAACGCTCCGATACCCATGCGGCAGCAACGTCCTTAGGAGCA
ATGAG
ACCAGTTGAAGCGCTGTACCAAATGCGAGCAAGAGCAAGAACGACGTTCCGCTC
GTCACC
CTTCCAATCCGACTCTGCATTCCACTGGGCAATAGTGTCGAAAAGCGCCTTGGAN
AAATG
CTCCTTCGGCNCCGGCTCGAAAAAC
Figure B .l l Alignment o f DNA sequence from figure B.10 with DNA from
gene bank.
Matched with aadA5
gb | EU9141 0 1 . 1 | E E s c h e r i c h i a c o l i s t r a i n 59 c l a s s 1
in t e g r o n , c o m p le te s e q u e n c e ;
e t h i d i u m b r o m id e a n d q u a t e r n a r y am m onium com p ou n d e x p o r t
p r o t e in ( q a c E d e l t a l ) , d ih y d r o p t e r o a t e s y n th a s e ty p e 1 S u l l
(su ll),
h y p o t h e t i c a l p r o t e i n , and ch r o m a te t r a n s p o r t p r o t e in
ChrA ( c h r A ) g e n e s , c o m p l e t e c d s ; a n d i n s e r t i o n s e q u e n c e I S 2 6 ,
p a r t i a l seq u en ce
Length=4828
S co re = 1003 b i t s ( 5 4 3 ) , E x p e c t = 0 .0
I d e n t i t i e s = 5 4 9 / 5 5 3 (99%), G aps = 1 / 5 5 3 (0%)
S tr a n d = P lu s /M in u s
Q uery 1 AGCCNGCCTTTC-
TGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAA 59
Sbjct 1730
AGCCAGCCTTTCATGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAA
1671
230
Q uery 60
TAATAAAAGCAGACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCT
119
Sbjct 1670
1611
Q uery 120
AACGCATAGTTGAGCGGCGGGCGCAGCCCGTCCGCTTGAACGCCGAGTTAGGCATCAGAT
179
Sbjct 1610
AACGCATAGTTGAGCGGCGGGCGCAGCCCGTCCGCTTGAACGCCGAGTTAGGCATCAGAT
1551
Q uery 18 0
GCCCTCGGCGCGGGTCGATGCACTTTTCGCACATGCCGCTCAACGCAAGATTCTCTCAAT
239
Sbjct 1550
GCCCTCGGCGCGGGTCGATGCACTTTTCGCACATGCCGCTCAACGCAAGATTCTCTCAAT
1491
Q uery 24 0
CGTTGCTTTGGCATATCGAACGAACGCGGCCGTCTCTTCGACGCGCATTGCTAGGTCGTC
299
Sbjct 1490
CGTTGCTTTGGCATATCGAACGAACGCGGCCGTCTCTTCGACGCGCATTGCTAGGTCGTC
1431
Q uery 3 00
GTCCTCGCTACCCAGGTACGCCGCGCGTGCCTTGCAGATGAGGGGCCGATGCTCGGCAGG
359
Sbjct 1430
GTCCTCGCTACCCAGGTACGCCGCGCGTGCCTTGCAGATGAGGGGCCGATGCTCGGCAGG
1371
Q uery 3 60
CAAACGCTCCGATACCCATGCGGCAGCAACGTCCTTAGGAGCAATGAGACCAGTTGAAGC
419
Sbjct 1370
CAAACGCTCCGATACCCATGCGGCAGCAACGTCCTTAGGAGCAATGAGACCAGTTGAAGC
1311
Q uery 42 0
GCTGTACCAAATGCGAGCAAGAGCAAGAACGACGTTCCGCTCGTCACCCTTCCAATCCGA
479
231
Sbjct 1310
GCTGTACCAAATGCGAGCAAGAGCAAGAACGACGTTCCGCTCGTCACCCTTCCAATCCGA
1251
Q uery 4 80
CTCTGCATTCCACTGGGCAATAGTGTCGAAAAGCGCCTTGGANAAATGCTCCTTCGGCNC
539
Sbjct 1250
CTCTGCATTCCACTGGGCAATAGTGTCGAAAAGCGCCTTGGAGAAATGCTCCTTCGGCAC
1191
Q uery 54 0 CGGCTCGAAAAAC 552

II I I I I I I I I I I I
Sbjct 1190 CGGCTCGAAAAAC 117 8
Figure B.12 DNA sequence from K. pneumoniae AES 135 (lkb) amplified
VAF and QacR primer
CNGCCTTTCNGATATATCTCCCAATTTGTGTAGGGCTTATTAT
GCACGCTTAAAAATAATAAAAACAGACTTGACCTGATAGTTTGGCTGTGAGCAAT
TATGT
GCTTAGTGCATCTAACGCCGCTATCAATTGCGGTAAAAAGCGTAGTGAGCGCGGC
GAACG
AAGCTTTTTGCCGTCAATTGCATAGCTTTGTTAACCCTTTTTCCAAATTTGATAGC
AATA
GTTAATGTTTGAACTAAAATGTTGCTCAAAAACAACTTCNAAGAAGTTGGGAATA
TTCGG
GAAGAAAACATCCCCTTCTGGCTCAATGTCNATCGTCGATACNTGGAGCGTAGAG
GCCAT
GGGCAACGTTTCTCTGTAAATCTCCCCGCCACCAGACACTATAACGTGACCGGNG
ANNNN
NNNTAGACCGCCCATGGCCTCTTCGATCGACGGGAATNCTACTACGTTGTCNTTA
TTGGC
CGNCCANGCTGANCGAGTAACNNCCGNNNATTTCCTATTGGGGAGNGCCCCCNNT
GATNN
NNANNNNTTGCGGNCNNNCAN
Figure B.13 Alignment o f DNA sequence from fig. B12with DNA from gene
bank
Matched with dfrA30
> g b 1J N 1 2 1 3 8 4 . 1 | A c i n e t o b a c t e r b a u m a n n ii s t r a i n RUH875
a n tib io tic resista n ce islan d
A baR 21, p a r t i a l s e q u e n c e
Length=1789
Score = 534 b i t s ( 2 8 9 ) , E xpect = 2e-148

I d e n t i t i e s = 3 5 1 / 3 8 6 ( 9 1 % ) , G a p s = 5 / 3 8 6 (1%)
Strand=Plus/M inus
232
Q uery 13 AGCCNGCCTTTC-
TGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAA 71
Sbjct 1044
AGCCAGCCTTTCATGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAA
985
Q uery 72
TAATAAAAACAGACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCT
131
Sbjct 984
925
Q uery 13 2
AACGCCGCTATCAATTGCGGTAAAAAGCGTAGTGAGCGCGGCGAACGAAGCTTTTTGCCG
191
Sbjct 924
AACGCCGTTATCAATTGCGGTAAGAAGCGTAGCAAGCGAAGCGAACGAAGCTTTTTACCG
865
Q uery 192
TCAATTGCATAGCTTTGTTAACCCTTTTTCCAAATTTGATAGCAATAGTTAATGTTTGAA
251
Sbjct 864
TCAATTGCATAGCTTTGTTAACCCTTTTGCCAAATTTGATAGCAATAGTTAATGTTTGAG
80 5
Q uery 2 52 CTAAAATGTTGCTCAAAAACAACTTCGAAGA-
ANTTGGGAATAT T CGGGAAGAAAACAT C 3 1 0
Sbjct 804 CTAAAGTGTTGCTCAAAAACAACTTCGAAGGTA-

TTGGGAATATTCGGAAAGAAAACATC 74 6
Q uery 311 CCCTTCTGGCTCAATGTCGATCGNNNATACATGNANCGTANAGGNCC-

TGGNNAACGTTT 3 6 9
Sbjct 74 5 TCCTTCCGGCTCAATATCAATCGTCGATATATGGAGCGTAGAGG-
CCATGGGCAATGTTT 6 87
Q uery 370 CTCTGNAAATCTCCCCGCCNCCAGAC 3 95

Mill I II IIII IIIII I M I N I
Sbjct 686 CTCTGTAAATCTCCCCGCCACCAGAC 661
233
Figure B.14 DNA sequence from K. pneumoniae AES48 amplified by QacR
primer
(1.5 kb)
GCCNGCCTTTCNGATATATCTCCCNATTTGTGTAGGGCTTATTAT
GCACGCTTAAAAATAATAAAAGCAGACTTGACCTGATAGTTTGGCTGTGAGCAAT
TATGT
GCTTAGTGCATCTAACGCCGGAGTTAAGCCGCCGCGCGTAGCGCGGTCGGCTTGA
ACGAA
TTGTTAGACATCATTTACCAACTGACTTGATGATCTCGCCTTTCACAAAGCGAATA
AATT
CTTCCAAGTGATCTGCGCGTGAGGCCAAGTGATCTTCTTTTTGTCCCAGATAAGCT
TGCT
TAG CTTCAAGTAAGACGGGCTGATACTGGGCAGGTAGGCGTTTTATTGCCCAGTC
GGCAG
CGACATCCTTCGGCGCGATTTTGCCGGNTATTGCGCTGTACCAAATGCGGGACAA
CGTAA
GCACTACATTTCGCTCATCGCCGGCCCAGTCGGGCTGCGAGTTCCATAGCTTCAA
GGTTT
CCCTCANCGCCTCNAATANATCCTGTTCAGGAANCGGGTCAAAGAATTCCTCCGN
TGCCG
GACCTACCNAGG
Figure B.15 Alignment o f DNA sequence from fig. B.14 with DNA from
gene bank.
Matched with aadA2
d b j | A P 0 1 2 2 08 . 1 1 1 3 E s c h e r i c h i a co li p l a s m i d pN D M -l_D ok01
DNA, c o m p l e t e s e q u e n c e ,
s t r a i n : NDM-1 D o k Ol
L e n g t h = 1 9 5 5 60
Score = 950 b i t s ( 5 1 4 ) , E x p e c t = 0 .0
I d e n t i t i e s = 5 2 7 / 5 3 8 (98%), G aps = 1/538 (0%)
S tr a n d ^ P lu s /M in u s
Q uery 1 GCCNGCCTTTC-
NGATATATCTCCCNATTTGTGTAGGGCTTATTATGCACGCTTAAAAAT 59
Sbjct 115050
GCCAGCCTTTCATGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAAT
114991
Q uery 60
AATAAAAGCAGACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCTA
119
Sbjct 114990
114931
Q uery 12 0
ACGCCGGAGTTAAGCCGCCGCGCGTAGCGCGGTCGGCTTGAACGAATTGTTAGACATCAT
179
234
Sbjct 114930
114871
Q uery 18 0
TTACCAACTGACTTGATGATCTCGCCTTTCACAAAGCGAATAAATTCTTCCAAGTGATCT
239
Sbjct 114870
114811
Q uery 24 0
GCGCGTGAGGCCAAGTGATCTTCTTTTTGTCCCAGATAAGCTTGCTTAGCTTCAAGTAAG
299
Sbjct 114810
114751
Q uery 3 00
ACGGGCTGATACTGGGCAGGTAGGCGTTTTATTGCCCAGTCGGCAGCGACATCCTTCGGC
359
Sbjct 114750
114691
Q uery 360
GCGATTTTGCCGGNTATTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGC
419
Sbjct 114690
GCGATTTTGCCGGTTATTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGC
114631
Q uery 42 0
TCATCGCCGGCCCAGTCGGGCTGCGAGTTCCATAGCTTCAAGGTTTCCCTCANCGCCTCN
479
Sbjct 114630
TCATCGCCGGCCCAGTCGGGCTGCGAGTTCCATAGCTTCAAGGTTTCCCTCAGCGCCTCG
114571
Q uery 4 80
AATANATCCTGTTCAGGAANCGGGTCAAAGAATTCCTCCGNTGCCGGACCTACCNAGG 53 7
Sbjct 114570
AATAGATCCTGTTCAGGAACCGGGTCAAAGAATTCCTCCGCTGCCGGACCTACCAAGG
114513
> g b 1HQ7 3 0 1 2 0 . 1 | EO E s c h e r i c h i a c o l i strain WM31a01

i n s e r t io n seq u en ce IS26, r e s o lv a s e
235
(tnpR) g e n e , and tr a n s p o s o n T n l 7 2 1 , c o m p le te s e q u e n c e ;
TnpM g e n e , c o m p l e t e c d s ; a n d c l a s s 1 i n t e g r o n , p a r t i a l seq u en ce
Length=4988
Score = 950 b i t s ( 5 1 4 ) , E xpect = 0 .0

I d e n t i t i e s = 5 2 7 / 5 3 8 ( 98 %) , G ap s = 1 / 5 3 8 (0%)
Strand=Plus/M inus
Q uery 1 GCCNGCCTTTC-
NGATATATCTCCCNATTTGTGTAGGGCTTATTATGCACGCTTAAAAAT 59
Sbjct 4710
GCCAGCCTTTCATGATATATCTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAAT
4651
Q uery 60
119
Sbjct 4650
4591
Q uery 12 0
179
Sbjct 4590
4531
Q uery 18 0
239
Sbjct 4530
4471
Q uery 24 0
299
Sbjct 4470
4411
Q uery 3 00
359
Sbjct 4410
4351
236
Q uery 3 60
GCGATTTTGCCGGNTATTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGC
419
Sbjct 4350
GCGATTTTGCCGGTTATTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGC
4291
Q uery 42 0
TCATCGCCGGCCCAGTCGGGCTGCGAGTTCCATAGCTTCAAGGTTTCCCTCANCGCCTCN
479
Sbjct 4290
TCATCGCCGGCCCAGTCGGGCTGCGAGTTCCATAGCTTCAAGGTTTCCCTCAGCGCCTCG
4231
Q uery 4 80
AATANATCCTGTTCAGGAANCGGGTCAAAGAATTCCTCCGNTGCCGGACCTACCNAGG 53 7
Sbjct 4230
AATAGATCCTGTTCAGGAACCGGGTCAAAGAATTCCTCCGCTGCCGGACCTACCAAGG
4173
Figure B.16 DNA sequence from K. pneumoniae AES48 amplified by VAF

primer
(1.5 kb)
NNNNNNNNNCNNNNNNCACTGNNNNNNNCTTGACCGAACGCAGCGGTGGTAACG
GCGCAG
TGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCAT
CCA
AGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGA
TGTTA
CGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATATGAACTCGGAATCAGT
ACGCAT
TTATCTCGTTGCTGCGATGGGAGCCAATCGGGTTATTGGCAATGGTCCTAATATCC
CCTG
GAAAATTCCGGGTGAGCAGAAGATTTTTCGCAGACTCACTGAGGGAAAAGTCGTT
GTCAT
GGGGCGAAAGACCTTTGAGTCTATCGGCAAGCCTCTACCGAACCGTCACACATTG
GTAAT
CTCACGCCAAGCTAANTACCGCGCCACTGGNTGCGTAGTTGTTTCAACGCTGTCG
CACGC
TATCGCTTTGGCATCCGAACTCGGNAATGAANTCTNCGTCNNGGGNGGAGCNGAG
NNANA
NACTCTGGCACTACCT
Figure B.17 Alignment o f DNA sequence from fig. B.16 with DNA
sequences from gene bank. Matched with dfrA12 dihydrofolate reductase
> >EM_PRO: DQ39 0 4 5 4 ; D Q 3 9 0 4 5 4 E s c h e r ic h ia c o l i s t r a i n 517- (63946 n t)

r e v -c o m p i n i t n : 25 62 i n i t l : 2562 o p t: 2562 Z - s c o r e : 2828.1 b i t s : 540.0
237
E () : 7 . 4 e - 1 5 1
b a n d e d S m it h - W a t e r m a n s c o r e : 2 5 6 2 ; 97. 5% i d e n t i t y ( 97 .5 % s i m i l a r ) in 528
n t o v e r la p (5 5 6 -2 9 :2 5 1 4 6 -2 5 6 7 3 )
550 540 530

-S e q u e - AGGTAGTGCCAGAGTNTNTNNCTCNGCTCC
fEM_PRO TACCTCAGATAGAAACACGCCGTGGGCGTGAGGTAGTGCCAGAGTGTATATCTCAGCTCC
25120 25130 25140 25150 25160 25170
520 510 500 490 480 470

Seque- NCCCNNGACGNAGANTTCATTNCCGAGTTCGGATGCCAAAGCGATAGCGTGCGACAGCGT
EM_PRO GCCCGCGACGTAGAGTTCATTGCCGAGTTCGGATGCCAAAGCGATAGCGTGCGACAGCGT
25180 25190 25200 25210 25220 25230
460 450 440 430 420 410

Seque- TGAAACAACTACGCANCCAGTGGCGCGGTANTTAGCTTGGCGTGAGATTACCAATGTGTG
EM_PRO TGAAACAACTACGCAGCCAGTGGCGCGGTAGTTAGCTTGGCGTGAGATTACCAATGTGTG
25240 25250 25260 25270 25280 25290
400 390 380 370 360 350

:S e q u e - ACGGTTCGGTAGAGGCTTGCCGATAGACTCAAAGGTCTTTCGCCCCATGACAACGACTTT
EM_PRO ACGGTTCGGTAGAGGCTTGCCGATAGACTCAAAGGTCTTTCGCCCCATGACAACGACTTT
25300 25310 25320 25330 25340 25350
340 330 320 310 300 290

Seque- TCCCTCAGTGAGTCTGCGAAAAATCTTCTGCTCACCCGGAATTTTCCAGGGGATATTAGG
EM_PRO TCCCTCAGTGAGTCTGCGAAAAATCTTCTGCTCACCCGGAATTTTCCAGGGGATATTAGG
25360 25370 25380 25390 25400 25410
280 270 260 250 240 230

Seque- ACCATTGCCAATAACCCGATTGGCTCCCATCGCAGCAACGAGATAAATGCGTACTGATTC
EM_PRO ACCATTGCCAATAACCCGATTGGCTCCCATCGCAGCAACGAGATAAATGCGTACTGATTC
25420 25430 25440 25450 25460 25470
220 210 200 190 18 0 1 70

Seque- CGAGTTCATATGGCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTG
EM_PRO CGAGTTCATATGGCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTG
25480 25490 25500 25510 25520 25530
160 150 140 130 120 110

Seque- CTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGC
EM_PRO CTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGC
25540 25550 25560 25570 25580 25590
100 90 80 70 60 50
S e q u e - ATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCA
EM_PRO ATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCA
25600 25610 25620 25630 25640 25650
40 30 20 10
Seque- CCGCTGCGTTCGGTCAAGNNNNNNNCAGTGNNNNNNGNNNNNNNNN
238
EM_PRO CCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTAC
25660 25670 25680 25690 25700 25710
> >EM PRO: EU 7 8 0 0 1 3 ; E U 7 8 0 0 1 3 K l e b s i e l l a p n e u m o n ia e s t r a i n (37606 n t)

i n i t n : 2562 i n i t l : 2562 o p t: 2562 Z -score: 2831.4 b i t s : 539.8 E():
8 . l e - 151
b a n d e d S m it h - W a t e r m a n s c o r e : 2 5 6 2 ; 97 . 5% i d e n t i t y ( 9 7. 5 % s i m i l a r ) i n 5 2 8
n t o v e r la p ( 2 9 - 5 5 6 : 8 3 4 3 - 8 8 7 0 )
10 20 30 40 50
Sequen NNNNNNNNNCNNNNNNCACTGNNNNNNNCTTGACCGAACGCAGCGGTGGTAACGGCGC
EM_PRO GCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGC
8320 8330 8340 8350 8360 8370
60 70 80 90 100 110
S e q u e n AGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATC
EM_PRO AGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATC
8380 8390 8400 8410 8420 8430
120 130 140 150 160 170

S e q u e n CAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGT
EM_PRO CAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGT
8440 8450 8460 8470 8480 8490
180 190 200 210 220 230

S e q u e n TACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATATGAACTCGGAATCAGTACGC
EM_PRO TACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGCCATATGAACTCGGAATCAGTACGC
8500 8510 8520 8530 8540 8550
240 250 260 270 280 290

S e q u e n ATTTATCTCGTTGCTGCGATGGGAGCCAATCGGGTTATTGGCAATGGTCCTAATATCCCC
EM_PRO ATTTATCTCGTTGCTGCGATGGGAGCCAATCGGGTTATTGGCAATGGTCCTAATATCCCC
8560 8570 8580 8590 8600 8610
300 310 320 330 340 350

S e q u e n TGGAAAATTCCGGGTGAGCAGAAGATTTTTCGCAGACTCACTGAGGGAAAAGTCGTTGTC
EM_PRO TGGAAAATTCCGGGTGAGCAGAAGATTTTTCGCAGACTCACTGAGGGAAAAGTCGTTGTC
8620 8630 8640 8650 8660 8670
360 370 380 390 400 410

S e q u e n ATGGGGCGAAAGACCTTTGAGTCTATCGGCAAGCCTCTACCGAACCGTCACACATTGGTA
EM_PRO ATGGGGCGAAAGACCTTTGAGTCTATCGGCAAGCCTCTACCGAACCGTCACACATTGGTA
8680 8690 8700 8710 8720 8730
420 430 440 450 460 470

S e q u e n ATCTCACGCCAAGCTAANTACCGCGCCACTGGNTGCGTAGTTGTTTCAACGCTGTCGCAC
EM_PRO ATCTCACGCCAAGCTAACTACCGCGCCACTGGCTGCGTAGTTGTTTCAACGCTGTCGCAC
8740 8750 8760 8770 8780 8790
480 490 500 510 520 530

S e q u e n GCTATCGCTTTGGCATCCGAACTCGGNAATGAANTCTNCGTCNNGGGNGGAGCNGAGNNA
239
| EM_PRO GCTATCGCTTTGGCATCCGAACTCGGCAATGAACTCTACGTCGCGGGCGGAGCTGAGATA
| 8800 8810 8820 8830 8840 8850
i 540 550
:S e q u e n NANACTCTGGCACTACCT
| EM_PRO TACACTCTGGCACTACCTCACGCCCACGGCGTGTTTCTATCTGAGGTACATCAAACCTTC
| 8860 8870 8880 8890 8900 8910
Figure B.18 DNA sequence from K. pneumoniae AES74 amplified by CTX-

M-15 F primer
NNNNNNNNNNNNNNNGTGCCGCTGTATGCGCAACGGCGGACGTACAGCAAAAAC
TTGCCG
AATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAG
ATAATT
CGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGT
GATGG
CCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGC
NAGTTG
AGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAANNACGTCNN
TGGGA
CNATGTCNCTGGCTGANCTTANCGCGGCCGCGCTACAGTACANNNATAACGTGNN
GATGA
NNAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACN
GCTGG
GANANNAANNGNTCCNNCNCGACCGNACCNAGCCNACNTTAANNNNNGNNNTTC
CGGGCG
A TCCGNGTGNTACN AN TTCN GCTCG AG TAA TG G AG CN CACTCCG CG GA TTN N GN N
NATGG
G TA TCG CNTTTN NN TGA CN TCCA ACG GN CN CNN CTG GN GA ATTGN N TN AN N GG TG
NTNNN
NNTNNTNNAGCGNNCATNNNNNCNGNNNNGNNNNCNNC
Figure B.19 Alignment o f DNA sequence from fig. B.18 with DNA sequence
from gene bank
>>EM PRO:EU93 573 9 ; EU935739 E s c h e r ic h ia c o l i s t r a i n A pi ( 117536 n t )

; initn: 3785 initl: 3785 Opt: 3 785 Z-SCOre: 3 7 9 6 . 6 bits: 7 2 0 . 5 E( ) : 6 . 3 e - 2 0 5
(banded Smith-Waterman score: 3 7 8 5 ; 100.0% identity (100.0% similar) in 757 nt
(overlap ( 1 - 7 5 7 : 6 3 0 4 0 - 6 3 7 9 6 )
10 20 30
i Seq uen GACCAGAATCAGCGGCGCACGATCTTTTGG
(EM_PRO TGCCTTAGGTTGAGGCTGGGTGAAGTAAGTGACCAGAATCAGCGGCGCACGATCTTTTGG
6 3010 6 3020 63030 63040 63050 63060
j 40 50 60 70 80 90
{Sequ en CCAGATCACCGCGATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTATCCCCCAC
[EM_PRO CCAGATCACCGCGATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTATCCCCCAC
j 6 3070 63080 63 090 6 3100 63110 63120
I 100 110 120 130 140 150

[S eq uen AACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCGCTGCACCGGTGGTATTGCCTTTCAT
240
! EM_PRO AACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCGCTGCACCGGTGGTATTGCCTTTCAT
i 63 13 0 63140 63150 63160 63170 63180
\
| 160 170 180 190 200 210

| S eq u en CCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCAATGCTTTACCCAGCGTCAGATTCCG
IEM_PRO CCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCAATGCTTTACCCAGCGTCAGATTCCG
| 63 19 0 6 3 2 00 63210 63220 63230 63240
j 220 230 240 250 260 270

| S eq u en CAGAGTTTGCGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGAATGGCGGT
!EM_PRO CAGAGTTTGCGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGAATGGCGGT
63 25 0 6 3 260 6 3270 63280 63290 63300
280 290 300 310 320 330

S eq u en GTTTAACGTCGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGTCGGGCGAA
: EM_PRO GTTTAACGTCGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGTCGGGCGAA
63 31 0 6 3320 63330 63 340 63350 63360
340 350 360 370 380 390

j S eq u en CGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACGTTATC
!EM_PRO CGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACGTTATC
63370 63380 6 3390 6 3 40 0 63410 63420
400 410 420 430 440 450

IS eq u en GCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTT
EM_PRO GCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTT
63430 6 3440 6 3450 6 3460 63470 63480
460 470 480 490 500 510

S e q u en TTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAA
; EM_PRO TTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAA
6 34 9 0 6 3 500 63 51 0 6 3520 63530 63540
520 530 540 550 560 570

!S eq u en CAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCGATCACTTTACTGGT
EM_PRO CAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGT
6 3550 6 3560 6 3570 63580 63590 63600
580 590 600 610 620 630

;S eq u en GCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTT
j EM_PRO GCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTT
63 610 6 3620 63630 63640 63650 63660
640 650 660 670 680 690

i Seq uen AATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTG
| EM_PRO AATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTG
6 3670 6 3680 63690 63700 63710 63720
700 710 720 730 740 750

ISequ en TACGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGT
| EM_PRO TACGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGT
6 3730 63740 63750 63760 6 3770 63780
j
jSequen CGCCATC
241
1EM_PRO CGCCATCAGCGTGAACTGGCGCAGTGATTTTTTAACCATGGGATTCCTTATTCTGGAAGA
! 63790 6 38 0 0 63810 63820 63830 63840
Figure B.20 DNA sequence from K. pneumoniae AES 140 amplified by CTX-
M-15 F primer
NNNNNNNNNNNNNNTGTGCCGCTGTATGCGCAACGGCGGACGTACAGCAAAAAC
TTGCCG
AATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAG
ATAATT
CGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGT
GATGG
CCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGC
GAGTTG
AGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAA
TGGGA
CGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGC
GATGA
ATAAGCTGATTGCTCACGTTGGCGGCCCGGCTAGCGTCACCGCGTTCGCCCGACA
GCTGG
GAGACGAAACGTTCCNTCTCGACCGTACCGAGCCGACGTTAANNACCGCCNNNN
NGGGCG
ATCCGCGTGATACCNNTTCNNCTCGGGCANTGGCNCAAACTCTGCGGANNNTGAC
GCTGG
NNNNNNCATTNNNCGN
Figure B.21 Alignment o f &/<zctx-m-i5 gene from fig. B.20 with DNA
sequences from gene bank
:>>EM PRO;EU9 3 57 39 ; EU935739 E s c h e r ic h ia c o l i s t r a i n A p i ( 117536 n t )

| i n i t n : 3790 i n i t l : 3 790 o p t : 3 790 Z-SCOre: 3 8 1 5 . 4 b i t s : 7 2 4 . 0 E( ) : 5 . 6 e - 2 0 6
[banded S m ith-W aterm an s c o r e : 3 7 9 0 ; 100.0% i d e n t i t y (100.0% s i m i l a r ) in 758 n t
{ o v e r la p ( 1 - 7 5 8 : 6 3 0 4 0 - 6 3 7 9 7 )
10 20 30
S eq u en GACCAGAATCAGCGGCGCACGATCTTTTGG
EM_PRO TGCCTTAGGTTGAGGCTGGGTGAAGTAAGTGACCAGAATCAGCGGCGCACGATCTTTTGG
6 3010 6 3020 6 3030 63040 63050 63060
40 50 60 70 80 90
{S eq u en CCAGATCACCGCGATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTATCCCCCAC
i EM_PRO CCAGATCACCGCGATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTATCCCCCAC
63070 6 3080 63090 63100 63110 63120
100 110 120 130 140 150

\Seq uen AACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCGCTGCACCGGTGGTATTGCCTTTCAT
j EM_PRO AACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCGCTGCACCGGTGGTATTGCCTTTCAT
6 3130 6 3140 63150 6 3160 63170 63180
160 170 180 190 200 210

jSequen CCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCAATGCTTTACCCAGCGTCAGATTCCG
jEM_PRO CCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCAATGCTTTACCCAGCGTCAGATTCCG
| 63 190 6 3200 63210 63220 63230 63240
242
j 220 230 240 250 260 270
ISeq u en CAGAGTTTGCGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGAATGGCGGT
| EM_PRO CAGAGTTTGCGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGAATGGCGGT
j 63250 63260 63270 63280 63290 63300
| 280 290 300 310 320 330

jS eq u en GTTTAACGTCGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGTCGGGCGAA
| EM_PRO GTTTAACGTCGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGTCGGGCGAA
{ 63310 63320 6 3 330 63340 63350 63360
340 350 360 370 380 390

[S eq u en CGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACGTTATC
| EM_PRO CGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACGTTATC
63370 6 3 380 63390 63400 63410 63420
400 410 420 430 440 450

;S eq u en GCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTT
EM_PRO GCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTT
63430 6 3440 6 3 450 6 3460 63470 63480
460 470 480 490 500 510

S e q u en TTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAA
jEM_PRO TTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAA
i 63490 6 3 500 6 3 510 6 3520 63530 63540
520 530 540 550 560 570

; S eq u en CAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGT
i EM_PRO CAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGT
63 5 5 0 6 3560 6 3570 63580 63590 63600
I 580 590 600 610 620 630

j Seq u en GCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTT
[EM_PRO GCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTT
j 63 610 63620 6 3630 63640 63650 63660
640 650 660 670 680 690

j S eq uen AATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTG
EM_PRO AATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTG
6 3670 6 3680 6 3690 63700 63710 63720
700 710 720 730 740 750

S eq uen TACGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGT
EM_PRO TACGTCCGCCGTTTGCGCATACAGCGGCAGACTTCCTAACAACAGCGTGACGGTTGCCGT
63730 6 3740 6 3750 63760 63770 63780
S eq uen CGCCATCA
EM_PRO CGCCATCAGCGTGAACTGGCGCAGTGATTTTTTAACCATGGGATTCCTTATTCTGGAAGA
63790 63800 63810 63820 63830 63840
243
Figure B.22 DNA sequence of ISEcpl from K. pneumoniae AES 140
NNNNNNNNNNNNANNAGCAGTCTANNNNNNNNNNNNNTANNNNNNTTTGAAGC
TAATAAA
AAACACACGTGGAATTTAGGTTTCATTCTGGCGACGTCCGTATTNGCCTTTCGGAA
GCAT
AAAATCGGACGCGTTGTGGCTCGCTTCAGGTAAAATATTGACTATTCNNGTTGTT
GTTAT
TTCGTCTCTTCCAGAATAAGGAATCCCATGGTTAAAAAATCACTGCGCCAGTTCA
CGCTG
ATGGCGACGGCAACCGTCACGCTGTTGTTAGGAAGTGTGCCGCTGTATGCGCAAA
CGGCG
GACGTACAGCAAAAACTTGCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGT
GTGGCA
TTGATTAACACAGCAGATAATTCGCAAATACTTTATCGTGCTGATGAGCGCTTTGC
GATG
TGCAGCACCAGTAAAGTGATGGCCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGC
GAACCG
AATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTATAATC
CGATT
GCGGAAAAGCACGTCAATGGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCG
CTACAG
TACAGCGATAACNNNNNNAAAAAAAANNNANAAAANNNNNNNNNNTGNNNNNN
NNCNGGG
gene bank
Matched with ISEcpl
>>EM PRO: EU93 5 7 4 0 ; EU935740 E s c h e r ic h ia c o l i s t r a i n C p i (93732 n t)

ir e v -c o m p i n i t n : 4018 i n i t l : 3840 o p t : 4197 Z - s c o r e : 3 8 8 5 .7 b i t s : 7 3 6 .9 E()
7 . le -2 1 0
;banded Sm ith-W aterm an s c o r e : 4 1 9 7 ; 96.8% i d e n t i t y (96.8% s i m ila r ) i n 893 n t
Io v e r la p ( 8 9 5 - 3 : 7 0 8 1 - 7 9 6 7 )
900 890 880 870

seq u e- TATTTTTTNNTTGNTNNNAAAGTTTGANTTCCTTN
EM_PRO TGTTTCAAATGATGATGCTTTCATATAACCTATTTTTGTTGTTCAAGTTTGA-TTCCTT -
7060 7070 7080 7090 7100
860 850 840 830 820 810

S eq ue - GGANTNNTTTCAGAATACAGACAGCAAATAAAGACCTTTCGTTTGAAGGTATGTATTTCT
EM_PRO GGACTC - - TTCAGAATACAGACAGCAAATAAAGACCTTTCGTTTGAA-GTATGTATTTCT

7110 7120 7130 7140 7150 7160
800 790 780 770 760 750

;s e q u e - TGCAGCAAAAAATAATCAAAACCGCAAGATATGTAATCATGAAGTTGTCGGAAAACTATC
t EM_PRO TGCAGC- AAAAATAATCAAAACCGCAAGATATGTAATCATGAAGTTGTCGGAAAACTATC

7170 7180 7190 7200 7210 7220
740 730 720 710 700 690

s e q u e - CGTACAAGGGAGTGTATGAAAAATGTCTGGTATAATAAGAATATCATCAATAAAATTGAG
| EM_PRO CGTACAAGGGAGTGTATGAAAAATGTCTGGTATAATAAGAATATCATCAATAAAATTGAG
7230 7240 7250 7260 7270 7280
680 670 660 650 640 630
244
IS eq u e - TGTTGCTCTGTGGATAACTTGCAGAGTTTATTAAGTATCATTGCAGCAAAGATGAAATCA
| EM_PRO TGTTGCTCTGTGGATAACTTGCAGAGTTTATTAAGTATCATTGCAGCAAAGATGAAATCA
j 7 290 7300 7310 7320 7330 7340
620 610 600 590 580 570

IS eq u e - ATGATTTATCAAAAATGATTGAAAGGTGGTTGTAAATAATGTTACAATGTGTGAGAAGCA
jEM_PRO ATGATTTATCAAAAATGATTGAAAGGTGGTTGTAAATAATGTTACAATGTGTGAGAAGCA
7350 7360 7370 7380 7390 7400
I 560 550 540 530 520 510

(s e q u e - GTCTAAATTCTTCGTGAAATAGTGATTTTTGAAGCTAATAAAAAACACACGTGGAATTTA
| EM_PRO GTCTAAATTCTTCGTGAAATAGTGATTTTTGAAGCTAATAAAAAACACACGTGGAATTTA
7410 7420 7430 7440 7450 7460
500 490 480 470 460 450

jS eq u e - GGTTTCATTCTGGCGACGTCCGTATTTGCCTTTCGGAAGCATAAAATCGGACGCGTTGTG
j EM_PRO GGTTTCATTCTGGCGACGTCCGTATTTGCCTTTCGGAAGCATAAAATCGGACGCGTTGTG
7470 7480 7490 7500 7510 7520
440 430 420 410 400 390

| s e q u e - GCTCGCTTCAGGTAAAATATTGACTATTCATGTTGTTGTTATTTCGTCTCTTCCAGAATA
| EM_PRO GCTCGCTTCAGGTAAAATATTGACTATTCATGTTGTTGTTATTTCGTCTCTTCCAGAATA
7530 7540 7550 7560 7570 7580
380 370 360 350 340 330

j S eq u e - AGGAATCCCATGGTTAAAAAATCACTGCGCCAGTTCACGCTGATGGCGACGGCAACCGTC
EM_PRO AGGAATCCCATGGTTAAAAAATCACTGCGCCAGTTCACGCTGATGGCGACGGCAACCGTC
7590 7600 7610 7620 7630 7640
320 310 300 290 280 270

S eq u e - ACGCTGTTGTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGACGTACAGCAAAAACTT
EM_PRO ACGCTGTTGTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGACGTACAGCAAAAACTT
7650 7660 7670 7680 7690 7700
260 250 240 230 220 210

S eq ue - GCCGAATTAGAGCNNNNGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAGAT
; EM_PRO GCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAGAT
7710 7720 7730 7740 7750 7760
200 190 180 170 160 150

is e q u e - AATTCGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCNNNNNTAAAGTG
;EM_PRO AATTCGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTG
7770 7780 7790 7800 7810 7820
140 130 120 110 100 90

;S eq u e - ATGGCCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGA
:EM_PRO ATGGCCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGA
7830 7840 7850 7860 7870 7880
80 70 60 50 40 30
IS e q u e - GTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAAN
IEM_PRO GTTGAGATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAAT
7890 7900 7910 7920 7930 7940
20 10
S eq u e - GGGACGANGTCACNGGCNGAGCTAG
245
EM_PRO GGGACGATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCG
7950 7960 7970 7980 7990 8000

M-15 primer
GGGAGTGCGCGGCGCGCTAGCTCAGCCAGTGACATCGTCCCATTGA
CGTGCTTTTCCGCA
ATCGG ATT ATAGTT A AC A AGGTC AG ATTTTTT G ATCT C AACTCGCT G
ATTT AAC AG ATT C
GGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTT
ACTGGTGCTGCAC
ATCGC AAAGCGCTC AT C AGC ACGAT AAAGT ATTT GCGAATT AT CT G
CTGTGTTAATCAAT
GCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTT
TTTGCTGTACGTCC
GCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGG
TTGCCGTCGCCATC
AGC GT G AACTGGC GAGCT G ATTTT A
gene bank
Matched with 6 /u c tx -m -i 5
e m b 1F R 8 2 8 6 7 6 . 1 | E s c h e r i c h i a c o l i p l a s m i d pCTX913 tn p A g e n e ,
b la C T X -M -15 g e n e
a n d d e l t a tn p A g e n e ( p a r t i a l ) , i s o l a t e 913
L e n g th = 2 6 5 6
Score = 678 b i t s (3 6 7 ), E x p e c t = 0 .0
I d e n t i t i e s = 3 7 7 / 3 8 1 (9 9 % ), G ap s = 4 / 3 8 1 (1%)
Q uery 7 GCGCGG- CGCGCT-

AGCTCAGCCAGTGACATCGTCCCATTGACGTGCTTTTCCGCAATCG 64
S b jc t 1312
GCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTTTTCCGCAATCG
1253
Q uery 65
GATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACAGATTCGGTT
124
S b jc t 1252
GATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACAGATTCGGTT
1193
Q uery 12 5
CGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGCTGCACATCG
184
246
S b jct 1192
CGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGCTGCACATCG
1133
Q uery 18 5
CAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAATCAATGCCA
244
S b jc t 1132
CAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAATCAATGCCA
1073
Q uery 24 5
CACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCG
304
S b jc t 1072
CACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCG
1013
Q uery 3 05
TTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCG
364
S b jc t 1012
TTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCG
953
Q uery 3 65 TGAACTGGCG- AGCTGATTTT 3 84

llllllllll II l l l l l l l
S b jc t 952 TGAACTGGCGCAG- TGATTTT 933

M-15 primers
TGTTCTGTAGCGCGGCGCGCTAGCTCAGCCAGTGACATCGTCCCAT
TGACGTGCTTTTCC
GC AAT C GG ATT AT AGTT AAC AAGGT CAG ATTTTTT GATCT CAACT C
GCT G ATTT AAC AG A
TTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCAC
TTTACTGGTGCTG
C AC AT CGC AAAGCGCT CAT C AGC ACG AT AAAGT ATTT GCG AATT AT
CTGCTGTGTTAATC
AATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAA
GTTTTTGCTGTACG
TCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGA
CGGTTGCCGTCGCC
AT C AGCGT GAACTGGC AAA AAT GATTTTT A
247
gene bank
Matched with blacjx-u -\5
g b |J F 9 1 8 4 3 3 . 1 \ E s c h e r i c h i a c o l i i n s e r t i o n s e q u e n c e I S E c p l ,
p a r t ia l seq u en ce;
a n d i n s e r t i o n s e q u e n c e I S 2 6 c e f o t a x i m a s e (b la C T X -M -1 5 ) g e n e ,
p a r tia l cds
L e n g th = 8 0 8
I d e n t i t i e s = 3 8 4 / 3 9 1 (9 8 % ), G ap s = 3 / 3 9 1 (1%)
Q uery 1 TGTTCTGTAGCGCGG- CGCGCT-

AGCTCAGCCAGTGACATCGTCCCATTGACGTGCTTTT 58
S b jc t 500
TGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTTTT
441
Q uery 59
CCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACA
118
S b jc t 440
CCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACA
381
Q uery 119
GATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGC
178
S b jct 380
GATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGC
321
Q uery 179
TGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAA
238
S b jc t 320
TGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAA
261
Q uery 23 9
TCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTA
298
S b jc t 260
TCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTA
201
248
Q uery 2 99
CGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCG
358
S b jc t 200
CGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCG
141
Q uery 3 59 CCATCAGCGTGAACTGGCAAAAATGATTTTT 3 89
1 1 1 I I 11 I I 1 1 1 1 1 ! 1 1 1 I M I N I M
S b jc t 14 0 CCATCAGCGTGAACTGGCGCAG - TGATTTTT 111

M-15 primers
T GG AAGT AAT ACCTT GAAAGCGT GGT AT GCT GAAACT AT AT CAAAG

AAGCCAAATACGAC
AT GGCGGT GGGTC AT CTCTT GCTAAAGT CATTTT GGGCGA AT GAAG
CCGTGTTTCAAATG
AT GAT GCTTT CAT AT AACCTATTTTT GTTGTT C AAGTTT GATTCCTT G
GACTCTTCAGAA
T AC AG AC AGC AAAT AAAG ACCTTTCGTTT G AAGT AT GTATTT CTT G
CAGCAAAAATAATC
AAAACCGC AAG AT AT GT AAT CAT GA AGTT GTCGGAAAACT ATCCGT
AC AAGGG AGT GT AT
GAAAA AT GT CT GGT AT AAT AAG AAT AT CAT CAAT AAAATT GAGT GT
TGCTCTGTGGATAA
CTTGCCGAGTACTTACCTATCATTGCTGCAACCATGAAATCCCTATT
G ATTT AAT A AAAA
AT GATT G AAAGGCGGTT GT AAAT AAT GTT AC AAT GT GGG AG AAGC
AGTCTAAATTCTTCG
T G AAAT AGT G ATTTTT GAAGCTAAT AAAA AAC AC ACGTGG AATTT A
GGG ACT ATT CAT GT
T GTT GTT ATTTCGT ATCTTCC AG AAT AAGG AATCCC AT GGTTAAAA
AATCACTGCGCCAG
TTC ACGCT GAT GGCG ACGGC AACCGT CACGCT GTT GTT AGG AAGT G
TGCCGCTGTATGCG
CAAACGGCGGACGTACAGCAAAAACTTGCCGATTTAGAGCGGCAG
TCGGGAGGCAGACT G
GGT GT GGC AT GT GATT AAC ACGGC AG AT GATT CGC A A ATACT AT AT
CGTGCTGATGAGCG
CTTT GCG AT GT GC AGC ACC AGT AC AGT GAT GGCCGCGGCCGCG AT G
CT GAAAAG A AAT G A
AAACAAACCGATCTGTTAAATCCGCGAGTTGACACCCAAAATCCG
ACCTTGTGACTATGA
CTCCCCATCGTGAAAGTCGCCTTGTGACATGTTTTGCGTGAGCTAC
GCTGTCGCGCTATT
AC ACGTCCGCGGGGGTTTTTTTTTT ATTT A
249
gene bank
Matched with blaCjx-M-15
K l e b s i e l l a p n e u m o n ia e s t r a i n C 18 6 5 TEM-1 b e t a - l a c t a m a s e
(b la T E M -1)
g e n e , p a r t i a l c d s ; TnpR (tn p R ) g e n e , c o m p l e t e c d s ; i n s e r t i o n
s e q u e n c e I S E c p l , c o m p l e t e s e q u e n c e ; CTX-M -15 e x t e n d e d - s p e c t r u m
b e t a - l a c t a m a s e (b la C T X -M -1 5 ) a n d h y p o t h e t i c a l p r o t e i n
g e n e s , c o m p le t e c d s ; i n s e r t i o n s e q u e n c e IS 2 6 , c o m p le te
seq u en ce;
f l u o r o q u i n o l o n e a c e t y l a t i n g a m i n o g l y c o s i d e - ( 6 ' ) -N -
a c e ty ltr a n s fe r a s e
( a a c ( 6 ' ) - I b - c r ) g e n e , c o m p l e t e c d s ; a n d OXA-1
b e t a - l a c t a m a s e (b la O X A -1 ) g e n e , p a r t i a l c d s
L e n g th = 8 3 7 8
S c o r e = 1408 b i t s (7 6 2 ), E x p e c t = 0 .0
I d e n t i t i e s = 8 5 0 / 8 9 0 (9 6 % ), G ap s = 1 5 / 8 9 0 (2%)
S tr a n d = P lu s /P lu s
Q uery 17 AAAGCGTGGT- ATGCTG-

AAACTATATCAAAGAAGCCAAATACGACATGGCGGTGGGTCA 74
S b jc t 2486
AAAGCGTGGTAATGCTGAAAACTATATCAAAGAAGCCAAATACGACATGGCGGTGGGTCA
2545
Q uery 75
TCTCTTGCTAAAGTCATTTTGGGCGAATGAAGCCGTGTTTCAAATGATGATGCTTTCATA
134
S b jc t 2546
TCTCTTGCTAAAGTCATTTTGGGCGAATGAAGCCGTGTTTCAAATGATGATGCTTTCATA
2605
Q uery 13 5
TAACCTATTTTTGTTGTTCAAGTTTGATTCCTTGGACTCTTCAGAATACAGACAGCAAAT
194
S b jc t 2606
TAACCTATTTTTGTTGTTCAAGTTTGATTCCTTGGACTCTTCAGAATACAGACAGCAAAT
2665
Q uery 195
AAAGACCTTTCGTTTGAAGTATGTATTTCTTGCAGCAAAAATAATCAAAACCGCAAGATA
254
S b jc t 2666
AAAGACCTTTCGTTTGAAGTATGTATTTCTTGCAGCAAAAATAATCAAAACCGCAAGATA
2725
Q uery 2 55
TGTAATCATGAAGTTGTCGGAAAACTATCCGTACAAGGGAGTGTATGAAAAATGTCTGGT
314
250
S b jc t 2726
TGTAATCATGAAGTTGTCGGAAAACTATCCGTACAAGGGAGTGTATGAAAAATGTCTGGT
2785
Q uery 315
ATAATAAGAATATCATCAATAAAATTGAGTGTTGCTCTGTGGATAACTTGCCGAG - - TAC
372
S b jct 2786
ATAATAAGAATATCATCAATAAAATTGAGTGTTGCTCTGTGGATAACTTGCAGAGTTTA-
2844
Q uery 373
TTACCTATCATTGCTGCAACCATGAAATCCCTATTGATTTAATAAAAAATGATTGAAAGG
432
S b jc t 2 845 TTAAGTATCATTGCAGCAAAGATGAAAT - - C - AATGATTT -

ATCAAAAATGATTGAAAGG 2 9 0 0
Q uery 433
CGGTTGTAAATAATGTTACAATGTGGGAGAAGCAGTCTAAATTCTTCGTGAAATAGTGAT
492
S b jc t 2901
TGGTTGTAAATAATGTTACAATGTGTGAGAAGCAGTCTAAATTCTTCGTGAAATAGTGAT
2960
Q uery 4 93
TTTTGAAGCTAATAAAAAACACACGTGGAATTTAGGGACTATTCATGTTGTTGTTATTTC
552
S b jc t 2961
TTTTGAAGCTAATAAAAAACACACGTGGAATTTAGGGACTATTCATGTTGTTGTTATTTC
3020
Q uery 553
GTATCTTCCAGAATAAGGAATCCCATGGTTAAAAAATCACTGCGCCAGTTCACGCTGATG
612
S b jc t 3021
GTATCTTCCAGAATAAGGAATCCCATGGTTAAAAAATCACTGCGCCAGTTCACGCTGATG
3080
Q uery 613
GCGACGGCAACCGTCACGCTGTTGTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGAC
672
S b jc t 3081
GCGACGGCAACCGTCACGCTGTTGTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGAC
3140
251
Q uery 673
GTACAGCAAAAACTTGCCGATTTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATGT
732
S b jc t 3141
GTACAGCAAAAACTTGCCGAATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCAT - T
3199
Q uery 733
GATTAACACGGCAGATGATTCGCAAATACTATATCGTGCTGATGAGCGCTTTGCGATGTG
792
S b jc t 3200
GATTAACACAGCAGATAATTCGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTG
3259
Q uery 7 93 CAGCACCAGTACAGTGATGGCCGCGGCCGCGATGCTGAAAAGAAA-
TGAAAACAAACCGA 8 5 1
S b jc t 3 2 6 0 CAGCACCAGTAAAGTGATGGCCGCGGCCGCGGTGCTGAAGA-
AAAGTGAAAGCGAACCGA 3 3 1 8
Q uery 852 -TCTGTTAAATCCGCGAGTTGACACCCAAAA-TCCGACCTTGTGA-CTAT

898
l l l l l l l l l l l M I N I M I I I 1111 II III11111 I 1111
S b jc t 3319 ATCTGTTAAATCAGCGAGTTGAGATCAAAAAATCTGACCTTGTTAACTAT
3368
Figure B.30 DNA sequence from K. pneumoniae AES 1001 amplified by

CTX-M-15 primers
TAAAT GTT AT GT GT GAG AGC AGT CT AA ATTCTTCGT GAAAT AGT GA
TTTTTGAAGCTAAT
AA AAAAC AC ACGT GG A ATTT AGGG ACT ATT CAT GTT GTT GTT ATTT
CGTATCTTCCAGAA
TAAGGAATCCCATGGTTAAAAAATCACTGCGCCAGTTCACGCTGAT
GGCGACGGCAACCG
TC ACGCT GTT GTT AGG AAGT GT GCCGCT GT AT GCGC AAACGGCGG A
CGT AC AGC AAAAAC
TT GCCG AATT AG AGCGGC AGTCGGG AGGC AG ACTGGGT GT GGC AT
T GATT AAC AC AGC AG
ATAATTCGCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTG
CAGCACCAGTAAAG
TGATGGCCGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGA
ATCTGTTAAATCAGC
G AGTT GAG AT C AAA AAAT CT GACCTT GTT AACT AT AATCCG ATT GC
GGAAAAGCACGTCA
AT GGG AC GAT GT C ACT GGCTG AGCTT AGCGCGGCCGCGCT AC AGT A
CAGCGATAACGTGG
252
CT GGTT AAAT A AGCTT GTCTTTT GACCTTTCC ATT GACGGGTTTT CC
ACCCGACTAAAAT
TTC AAGCGC AAT ATTTTT ACTCC AACGATTT ACG AGT AGTT CTTT CC
TTTTTT C AAAG AA
CGCCGGGTCGGCCTTCATGGCGCTCCCACCCAATTGCCCACAAACT
ACCAAAAATTCGAA
TTTTT ACCCGTTT AAC AAT GAAGCC AACT GCCC ATCCCCCC ATTTT C
TACT GAT GTTTTT
TCTACCATCTCTTTCCTCACGCTGCTTTTTTTA
gene bank
Matched with 6/<3ctx -m - i 5
A c i n e t o b a c t e r b a u m a n n ii s t r a i n HI h y d r o x y i s o u r a t e h y d r o l a s e
gene,
c o m p le te c d s ; d is r u p t e d p y r im id in e u t i l i z a t i o n t r a n s p o r t e r
g e n e , p a r t ia l seq u e n c e ; in s e r t io n seq u en ce IS E cp l tr a n sp o sa se
(tn p A ) g e n e , c o m p l e t e c d s ; CTX-M15 (b laC T X -M 15) g e n e ,
c o m p le te c d s ; d is r u p t e d o r f4 7 7 g e n e , p a r t i a l se q u e n c e ;
tra n sp o so n
Tn3 tn p A g e n e , p a r t i a l s e q u e n c e ; a n d h y p o t h e t i c a l p r o t e i n
g e n e , c o m p le te c d s
L e n g th = 5 2 2 4
I d e n t i t i e s = 5 4 3 / 5 4 8 (9 9 % ), G ap s = 3 / 5 4 8 (1%)
S tr a n d = P lu s /P lu s
Q uery 9 ATGTGTGAG-
AGCAGTCTAAATTCTTCGTGAAATAGTGATTTTTGAAGCTAATAAAAAAC 67
S b jc t 2615
ATGTGTGAGAAGCAGTCTAAATTCTTCGTGAAATAGTGATTTTTGAAGCTAATAAAAAAC
2674
Q uery 68
ACACGTGGAATTTAGGGACTATTCATGTTGTTGTTATTTCGTATCTTCCAGAATAAGGAA
127
S b jc t 2675
ACACGTGGAATTTAGGGACTATTCATGTTGTTGTTATTTCGTATCTTCCAGAATAAGGAA
2734
Q uery 12 8
TCCCATGGTTAAAAAATCACTGCGCCAGTTCACGCTGATGGCGACGGCAACCGTCACGCT
187
S b jc t 2735
TCCCATGGTTAAAAAATCACTGCGCCAGTTCACGCTGATGGCGACGGCAACCGTCACGCT
2794
253
Q uery 188
GTTGTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGACGTACAGCAAAAACTTGCCGA
247
S b jc t 2795
GTTGTTAGGAAGTGTGCCGCTGTATGCGCAAACGGCGGACGTACAGCAAAAACTTGCCGA
2854
Q uery 24 8
ATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAGATAATTC
307
S b jc t 2855
ATTAGAGCGGCAGTCGGGAGGCAGACTGGGTGTGGCATTGATTAACACAGCAGATAATTC
2914
Q uery 3 08
GCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTGATGGC
367
S b jct 2915
GCAAATACTTTATCGTGCTGATGAGCGCTTTGCGATGTGCAGCACCAGTAAAGTGATGGC
2974
Q uery 3 68
CGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGAGTTGA
427
S b jct 2975
CGCGGCCGCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATCAGCGAGTTGA
3034
Q uery 42 8
GATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGAC
487
S b jc t 3035
GATCAAAAAATCTGACCTTGTTAACTATAATCCGATTGCGGAAAAGCACGTCAATGGGAC
3094
Q uery 4 88
GATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGCTGGTTA
547
S b jc t 3095
GATGTCACTGGCTGAGCTTAGCGCGGCCGCGCTACAGTACAGCGATAACGTGGC - GATGA
3153
Q uery 54 8 AATAAGCT 555

I M I N I
S b jc t 3154 A-TAAGCT 3160
254
Figure B.32 Alignment ofRpoB from K. pneumoniae AES817 sequence
type as ST 511 with gene bank
100 110 120 DO
-I
Rp«i nTW DJCS6DCTH3CCSBKTaW CC6CW HCB6aPiTTCani>CCglTICgW CtMDSSCTTRTDECSCSCW H61GCflflCCGCCIGflCETT6CflT6TTCBDCC£flTCflflIGCflCGGnG
KpoS817 TTTIIGCCKfiGCAGTRRCfCCGGKTCAACSSCflRCflGCfCGTTCCIITACCGGTKtflKtMiCGGCTTniXAGCGCGCflGfKl GGMCCGCCTGAC6TTGCfiTGTTCGClKttATC(WTGC8C(XTTG
Consensus TTTRGtXKGGCnETf)ROCCBGRGTCMCGGCMCnKf)CfilTCD)TnCCGGTKQncnCCGGCTTHTCnQCGCGCOOKl G GM CCGCCTGKGTTGOITGTTCSCfaxnianiGCnCGGTTS
131 140 160 170 180 220 230 240 250 2G0
I- 1 1 1
6C6TCHTC6TETTCQI£nnC6GGHTOK66flCGCflCCG8CliGflTiCQCCT6CTGGETGERTnCETlXRTSm6TCinC£TG6C6CGGCT6RflC(W6C!6fiflTTC6CCTTT6CTfC6GCflG6TflfC01
nrfiTnrrrtTCrTrrwnrtgnnrnH^HTrrBrsBnarrnrrTnrrofifiTH-innrriTTTnTf.TiKTrMrnMi s K i n a m » n n » n n a
Consensus BCfirt»raT6TTCDfiS*C06fflTnw^GCBCnaC6fflTflCaC£T6CI666T6GBTflCfiICCflTGTB6TD»CCT66 CGCGGCT6flflCfl86CT66flITCfittTTTGCT(COGCH56THflCa)
261 270 280 290 300 310 320 330 340 350 360 370 380 390
I- -I
Rp4 MTCTTCT8CGHR6T6HX61 1 1IC>lCC966nG6W
6TTCSCCTG86CSHTW
C6IKTrGttnCI1UMT9EIJW
C8GGTB6TG8BTnC6TC86TBBCCflDCgTTHflDCTnHCGBTBCfiG
RpuM17 S8TCTTmC6flt t i m X S I 11101 rtC966TT M KTTCSCCTS8GC6ITMC6Tll6nt t £ TTCTTCarraBC9MCfl66Tfl6T5MTTTC£TI^TT8CC9t9tt£TT66TCflCTTTnCSffr9C8G
Consensus 6flTCTTCT8CSHR6TUaXfil 111UlTCC8GGTTGGRGTTt6CtTGKCGAT8K6TKTTGCCTTCTTCSRTHGC8GRC86ETn6TGflRTTTCSTQI6TRaCC8C8(X6TTCETCHCTTTnCSBnES6
391 400 450 470 480 490 501
RpoB lb 11lUVttBtHXHIHI 11Jii TuulllifCbCbIHCHUiO«lB6b6flbII ft!ICflhHUSlIbTllSHtX111*1*111181118 IHblobGI

Rpo8817 tt tcTowsrecOTfirnXTTQGTCHiC8CfiTBCflC56fCfl66Gfl6TT8RTCBGflC£fifiTGTTlfiGflCrrrCHGGC6nTCfiflTCHiflCHTRCGCGRCTGI[B6T8G67
Consensus CBTCTOWGfififlGCCflTflrnXTTQSTCTIiC6C6TRCHCGGflCHG6fifl6TTfifiTCflGf(XGflTGTTCGGfi(XTTOCGCfirTTDjflTCt)ljflCflTRC5C6flCCBlrBGTfifiGT
Figure B.33 Alignment of GapA from K. pneumoniae AES817 sequence

1 10 20 30 40 50 60 70 80 90 100 U0 1 2 0 1 3 0
I............ . —» ....■■■»■ — »■ ■■ — i— ->■■ * ------- »........ 1 ---- 1--------1
IWCCTGRMiT6G68CEiWGrTG6T6TTGHCETTGTTGCT6aRECinCCS6T8TCTT(XTtMX68CGMMCC6CTCSTMMCBC)ITC8CCGCTG6CSC6MMMKTCGTTCTG8CTEGCCC6T(XMK
M
CCTSIM
6T666HC6H06TT66T6rt60CSn6nBCT6IW
6CiaCCS6T8TCTTCn68IXfiBC6HaHIXSCrC6TflHBCHOITOlCCSCT66C6C6W
WWH6TC6TTCT6flCT66CtX6TCCflHH6
mCCTGRMTGGGRCGmGnGET6TTGflC£TT6TTSCT6<n6CMCUi6TllTCnCCTGi)tt£flC£fW)tCGCTC6TflfncnCRTCOCC]jCT66UjC&ARflRflR6TCOrTCT&flCTGGC(XETCCI)HnG
131 260
I—
•C aO CK lCtS
R TB TTC STTC OnOC KTM CniSICKT TKtaiGtCaBGIinTC STrra
SM C SCTT IXTEaiCCaCtMCT ECC TiG C StCttT
S fiCTMK rn
iT ta
R CtOa M C TT--1
tStrar
iCll(C«CrCCEfllSTTC6ITCt>CG6C6CTH8CTTCCflC6CrrRCStTG6CC8G680IIC6TTTCa*C6£TTC£TG£8CC8CaWCICCtT6GCfiO:6CTG6CT(M)6TTHTCflflCfi8CflflCnCE6TRT
K M CICTCD8(T6TTtSnCfiCG6C6CTIiRCTTCSKfiCTTRCBCaG6CC8ESK8TCtTTTCCM CSCTTCtTEflCnHCDnCTGtCT6GCfitt6CTGfiCTIW RGnHTCIIRCSi)C>nCTTCG6TRT
261 390
Ml I— ... .............. .......... .................. ...... --1
nO *017 C6TT&AH66CCT60T«iC(llCCSTCQ)C6CTnCCICniCTRCTCHIMMCC£TT6RTGGCCC6TtTCflCIIIW68CTGGCGCG6(6GCC6CG6CSCflGCTO)GfnCATCnnXCErCCTCT0CC6GCfiCT
CSTreflBBCCCTCflTSflCIBaiTCaCSCTflCOCOTIRCTCIGIIflRRCtinfiflTGBCO^TCTCICIOWCRCTCfiCSCSGDjOCCSCfjGCSCneCTCflCflflCBTCflTCIXETCCTCTRCCGGCCCT
391 450
I— —I
h 4 SCTMKClKTRGSTmKTOCTGCCRGIICTGflflCEKMHCTBCCGSTRTEGCETTt
Consensus SCimGOCIKGIMEIIICIGCaGHCIBUCGGaillCliHCCESrNlBiCEITC
255
Figure B.34 Alignment of infB from K. pneumoniae AES817 sequence
l i d 20 30 40 50 60 70 80 90 100 118 120 130

|----- 1------ I------ 1------ ►
— H------I 1------ 1------ 1------ 1------ 1------ 1------ 1
inffl 6TCSTC6flTG0^rflCC(^TnC60iif¥^TGGft(3flflCTGSCICTCSCDmCflCT(Tr(XG6G«iGfiTSCaT#:TB6SfCnsnCfincnCfCfitfiftTC£fiGflTCGGCnCTG6CnRTCS
infB817 GT^CSRTGCIX&TflXCSCTTTCHSGHRnareaCGflffTGGCTCTCGtXGCCCOCTCnCCGGaiGGRTGCCGTfCIGGGiOGTTCGTTCTICflCGCGRTtXGfflTimTTCTGeCTTRTCG
Consensu GTC6T(^TGa^TIC(XI£TTTCSD£flBfCGTGQCfifllCTGGCTCTCBCt2CIXQCTCTTD^jSCftGGflT&C££TfCTffiGflCI*rnffincnCfCtiC£fiTCCGGflTCSBCnCTGGCTTRTCG
131 140 150 ISO 178 180 190 M 210 220 238 240 250 260
| , 1 . 1 1 1 1
iafi flTcnGTTCficcsccBCTRccfcaKnccTamGcmFBaTKTGGflTfKTTCBBTaGTaa^GCinacecceTcsTaBccscactmfieftfccncGfiTHTraTcscrrecficaxac
irrfB817 ^TCnGTTC}CO*CaCTICacajGT(CCTffiGCCKTn(KCSlB:TGGBTIBCTTCG8TBGTCTG(^GCflTClCfi(XBTOTCTGCISCaC13CaiGflflCGCSBTflTCCGTCBCCTGCBCSCaC
Consensus rcnGTTOI(IfiQJCTHCDCCG611C£TGC6(XSClTTfl6CSTGCTfiGflTflGClT(^TIIGTCTGCSGCfiT(SCSn:6TC5TCTSCCSCaiXfCCRGflflCOCBflTflTCCGTC6CtTSC6C6CaC
2S1 278 280 290 300 310 318
I----- 1------<------ 1------ 1------1---- 1
irtfl SflGCSCfiCSTGGSGfiTMRCGCGSCGTGGCCCGSGGTRTCCflGGRRGGTGRTCflTGCC
infB817 a « J C^ T6aBCTMCSCfiSCiTm ^ X Sm T lT C a B a ^ T g m a T g t
Consensus jRGDCfnTGBKSIflMCGCGGCEriSClXSGGGTBTCtnGlRliKTGRTCliTGlX
Figure B.35 Alignment ofPgi from A', pneumoniae AES817 sequence

1 16 20 36 40 56 86 76 80 SO 166 110 126 136
I----- 1------------ 1----- 1----- 1----- i----- 1
8RGTCCM
Pfi C6BTM C1BTETTS8CCSTRKfiGCC8CtCS6nCM
ni(IIW
TG6DXfM
n3ITCT6EfiETGRGCC&GSClD3VC86TDBCRCSCETTCTM
XR6CrG8TCQCCnGGDIX)M
I
3gli:n<CEtniCTTSTT6l<XET(<CfiHXICgE£TaBHCT8COMCT6£DI3OT3rCT8SS6T8MrrSBSCKO>fgTaKaCSgTTCT8Ca^SSTtaCOBSGCMIl<)6l
Pfiffl.7
Ummm 8W[aKSCTCTrtnyptiiMatcKg«»wrwTBrrriinrffCT88ttiaBag6aaMgTaB3TOTTni»xa6CT8aTcwrM««X8Mi
131 146 150 186 170 180 190 766 210 £20 230 240 250 280
I ►- > - i > 1
Pei lesiEttiHMTTartttTcceecTircEiteMcramrsEaKamjBiKTBtTeTnirncrTcscajBflraBscctTBficcnTETMiTiicEsaETBErTa
PeiU7 ^ J»fT n :T iy y T T T [w y T fT ttrm » ^ r f g « g fr « 7 y 'CTacg»IITtM BCTICT8TCT W JfCTTC8Cn3WCC8BSSCttT6BCrnTBETIWriXCSCfi88ETSErT81
TGGtSCt8TggiTTTOirCS[TII£grT8naCCtK3»IICSCT6TCrSKOI18TOPIW CT6CT8TCTIICrTCntSCa3BW CCfiH6SCttT6aiTTTGETIWjTDXEC888ETB6rT6l
281 276290290300 318 3 2 0 3 3 6 3 4 0 3 5 0 3 6 0 3 7 0 3 8 6 3 9 0
I--------- 1---------- i ---------- t ■ <■— - t .......... - t ---------- 1
SCa66S6TBTCaaTI^TaBBKn3Hmgl6EaEtHCSTKTSCEfiTTaW ^rrCS8R6ETW Ca3miEIW CTa3ITCCTStTEtSTSH6BTaimETTaKCTCfi666C8
GDIGSSRTHTCHXnTOtGtfiTiHHiXISGCSHCCtlGGRBDCGTSGTBXGTTCnRfST& rrCSRRGETfnCCGCCCSKTinCTCCaTCCrSCTECSTGRGRTQICCCCSTTCHGDCTCGGG& CS
8C8GSK18nX9rrC8G6STM I)EKlXfflCGHCttTG6R6CnQiTSGT6ClXTTClHfil8rTCS8ffiETfnCC&CCt8KT88CTIX8TCCr8CTECETSnGRTDCCCt£TTrfnXTCQGGGtS
CIMTCDgMKCP—CTinCECaBMP
256
Figure B.36 Alignment ofPhoE from K. pneumoniae AES817 sequence
1 1 9 20 30 46 50 GO 7V M 90 100 110 120 130

I 1------ 1
HmE T i T r f f i f i T T m T m ^ B i T T T n g m w w n r i m T r i r B y m r n r T w n r r ^
P M U 7 6Tl»U C£TanaKTgTMTTTim SBCKCEK n tm tT C K (a a ^ M C a^ lX E gC « Tltrm giTa«aCCTH Ê6C agSG C a^T T CaW«SCflH6a:T6G£
Consensu* 6T(XGCRCnCETT1IIKTHTERTTTlSHXGOBCGRCTTIXCCETDBCSCBGCniEIICCf^CIXRD3T1KQWBiTCBGnCCTGCT6GC)XGCGGCaKGETTCSnnKSGMDCTGGfi
131 140 150 180 170 110 190 200 211 220 230 240 SO 260
|------ 1------- 1------- «- .. . t------- 1------- 1------ 1
u cactsH rrB iH H iH T C H am w ^ T tT H gT M C H caT rK T C T 6aw n T iy»»B iin ^ iJscgim m 6caw 3> w gscaE fficin tflflG cssT 6E csaK T R ra
Ph«£8i7 ^"^vTnrîrrirrnrnTTTnrrnrinrnrnminrTMnffrrnrnflMTiariTniffrKritfmtttthtmm u wk m tnan.!Hiiwjijiiiiniin
Consensu* CSCQGCnBMTHTBCtXCflRanninKCTGGCGMXHTETM^CTGRRKaXIMIITGRCQXSRIDGCGGCGGCTTTfiCCiCMnGCSCflGRKTTTMGCG&TQGCGQCTRTCO
2£1 279 290 290 300 310 329 330 341 350 360 379 389 390
| 1- 1 1
ft*E tT T ea C T T tg T C T ta n m a ria g T B r tT E a C ia < W « S B ^ T in a a B 6 t f TM 6U gTM^CTgni«aiCHTCSICSTS6aiIE8CnKTlCTTaWCHHmRC«T6
2WEH7 6TnSKTTCfi8TCiaiID^lCgni8ariBreTB:T6TCO>WMBW EttT8TCaMBBStl68668ET8B8E8TCT(gni>CTICinCfiHCfiTSEait6W tITITTICTIDW
DWBHBOIT6
Consensu* rnrnnrnrrifiTrTB'iirrnTrrrTrwirTnTiiTH'TriTrMMirfifinniifoiTnirHiififfifiiiiK/infiTfiiiwflifrriiimwrTnriiTrmrriTrjriHTTniirrninrmiTi—wnriiTr.
391 400 410 420
I------, -------, -------1
PhcE flBCSCCTTGETSSBITflDWHHTDWCOK
PhoE817 HHCHXTTCSTGGHTTnOyMHTCflHCQS
Consensu* WCSCtTTCSTGEOTTnCJVUIlTDWCOC
Figure B.37 Alignment of tnoB from K. pneumoniae AES817 sequence

i l * 20 3 0 4 9 5 9 S 0 7 I B 0 9 0 100 11* 120130

I . . t t----- 1------ 1----- 1----- 1----- 1----- 1----- 1
tn d CTTCKn^lltaTKCnaHTGCGCfE[HrTGKQCG(HTKHTIfiGIIÎSIWCSCttCTnTCfiCCC8S£CCSCrT6GHBDfiTfMCGGT6SETTTKCC!iC[(CT(TCtflK15Cn
tnoffil? m i^lX firim T IC C T T niiT 6C fiD ttfiD :r»«C fiam M T (IM m m iC W rtfT(KCC6Cafi^n6S^fl(tffiTG(mT!KC5CrttHTC58ttT6CTT
( M M C n ilX g T K C T g T ltd n BM m C T Ig g g g c a B l B ^ IMK g a C TttT i m C g S a ^ ^
131 140 150 160 170 190 190 290 210 220 230 240 259 260
| 1 1 1—11 1
GGCGCr(TCSETBCECKTIT TE1ITSrTTTCRRRCGSCGffiGCCGBRCGCGGCrCrSCTGCCSKTTCICTTlira:rTC6SCTGTTa»CCTTTnCTCCG8CTI *5CTTS6BT rTfl6ETTT66
U rf817 GGCGCr&TRCSCSCCSKBXtTflTTSlrTETTnavnCGSCSRGSCCQSRCSCfiGCrCTbQISCCSSCTTCItTTCCCffnrSfiCTGTTCflftCCTTTnnCCfifiCTTRGGCTTSttTTTBKTTTGG
Consensu* 66CCCTiTB3CgCgegC6TITOTmt«ffiffl« B ^ ^ 11ntlClffiCTfflSSCnS6tTTTfl66TTT66
261 270 2*0 290 300 310 320 33# 340 350 360 370 300 390
| 1 i ■4 ■■-* —h----- 1------ 1----- 1------------ 1
-
tnd 6CTTIMnW6nCCSTTT*TGGIITIlXIX8GCHITCTnCGGCGGlTattaE[KCrCTEKTCtiGtTDBnCSS6TTCa(M6G6CTCaC6RD)GSCrSCtC£tCCG£RE6C66
tno8817 6(TrCSffinilttTTCMTTTflT66flT0IXMXS6(5CncrrnMGGrTtaGSDrTltCTCTSSn(I6fifT0ttTTI366TTt8(l(Mfi6OI(C6ftCf)G6CT5ttCCSCC66ft66Cfi6
Consensus P fK IM B M B M M B in W M ttlO W n W M an g llK B K IW n n M iM W M P B B B P g T B B IC T B H i
391 400 410 414

|----- ,-----H-|
Inal CTCMGRrCSGCCOtKIKCB!
tno6817 nCflfKATCGGtCSffSCaiCaiT
Consensus CTCMQITCGGCCttCttami
257
Figure B.38 Alignment ofM DH from A', pneumoniae AES817 sequence
1 1 9 20 » 40 GO 70 80 90 100 110 1 2 0 1 3 0
I ------ 1
NA817 &6C&CJ56HrGTB6TGCTGfiTCn;CI>CQGGC6T6GCBCGTHBGC(X8SCBTG&flTCSTTIX6HCCT6TTTHBT6TSHHTeC8&6TRTCSTBflHGnRCCIOjI6CfleCFI&flTTSCCaflHfCCTSCi:CGCfl6C
Consensus G8H^T6TKTQTTGRTOIXH^GH^ffiCSCSTRHBCIIfiGC8TGG^CGTTIXGMXTETTTffiTBTGRRTGC8BGTRTC£TGRHGffCCIC&T93)GCnGHTTGCCMNXTSCCCECI)GE
131 140 150 160 170 188 190 ZOO 210 220 230 240 250 260
I 1------1------ 1--------------------------->—....»■■■----1--------- 1
-
H i Cn6UTCSfiCflTTHTC(CDHClIE6TEaflTICQCtETESCTBTCgtfi(XSllHfiTIICTfiMWHM6IIfi6C6T6TICEBTHWMIClWCTGTTCEGCETTHCtl[g1ttttlTCHTIXSTTCCfl<l
HdhH7 CCT6CBTC6SCHrrHTCIMXWiXCS6TfiWnWXWIXSregTBTCfiCC6CCliHB6TtCreHHHIMWEOIttCST6nCHITHHHHHCflHBCT6TTCS6CSTraCCTCSCTSMaiTUWCt STTC£Bi
6 — — r r f f r iT r r f f in n T r n r f M r r r f f m m iif iif i iffi m i n i i n i m m i t u — i i i i i i i n n m u i i i — wuww 1
1m i n i m i n i m i i i i mi i i i i i m i i i m i
261 270 280 290 900 31( 320 330 340 350 X0 370 380 390
I ------ 1
Nil T(CCTTTGTGSCGCflKT6»l«lfiTW()TC6G£9(CC9XGT(;G^TCCC6GTCflTTGGTGCTC8CTCC6G66TCIICC8TTCTGC£TTT(CTGTCGCflGflTCCIXGGC6TD®CTTTfl6CGflTCflfiEflfl
HA817 TICCTTT6TGGC6G8KT6nfG6TMRTC&GCnCCGR66TG6H86TCtC66TC8TTGGT66IC8CTCC&GE6TCflCCilTTCTBXTTTICTGTC6C8GRTIXCCGGC6TC8GCTTTHGC6ilTCnGEflR
RCCTTT6TG6C8WTGffi8E6TiMTCG6CHRCC&RG6TG6H>)6TCCCG6TC8TTGETG6TCICTC£GGG6TCiX8TTnBITTTICT6TCEClGRTCCCCGBXTCR6CTTTnGC9ITCffi68R
391 400 410 420 430 440 450 460 470 477
Hdh HTTHXERdTGflaRHHCGTRT IDfcflHLbLLhtTROTHflEK bTfiGKulflHRGajGbCBGCBGbTCbKhRCLnbTCfiHTG

DM 17 flTT6CC6flCCI6flCT(MC6THT C86RflC6CC6£TflCTGRRGTI ST66MGC6flHR6t6G6C HG66TC66CSCCTT6TCSHTE
Consensus fllTSCC8CCTfiflCT(MCSrRTTCS6flflC6CC66TSCcfiflfl6TCSIS8»GCSflfiflGCffi8CfiGCfifi6TCfiGCSR(ITT6TCSflT6
Figure B.39 Alignment of mdh from K. pneumoniae AES809 sequence

1 1 0 2 0 3 0 4 0 5 6 6 0 7 ( 8 0 9 0 100 118 1 2 0 1 3 0
|....... t........ *------ 1----- 1------ 1
ndk (K6C6GIT6TBljT6CTG*TCTCCfiC6C6C6Ti6C8C6TflH6(XCB6CflT66flIC6TTCCfi8CCT6TTTIflr6T5flflTGC666TflIC6TGfiflS(KCICE16£8GC()GflTT6CDlRflflIT6CIt6Cfl6G
M faM El a a n a T 6 ]g T g T aT C T C tS C S 6 6 C 6 T K I^ T I» H X I^ !S 6 flri^ n iIS « I1 6TTTW 6 TS« TSai6ET H T aniC T C n CST6M6C<WTBECaM aCa6Ct t SCIK
Csmsmbs G6CSC^TBTa6r6aSBTCTq^CgBOTjg^TfBSCCCSBDjT6mTt^TTlXBHCCTSfrrTIRfST6tWT6Cj66THTCSTeflH6HfCrfC6TfrWinMf<<rrMMrrTrirrrnrnS6
131 149 150 160 170 180 190 200 aO 220 230 240 250 260
I...... ♦....... I----- I— ♦ ■■- I........ -I------ 1----—I.......1................ 1
nit rrTiran^TinrsmMfiTttjrHWBfrorttTrmanmfimaflfiMTHMonMfici^
.
NA8&9E1 CCT6mC6GC8mn«XA(ICCCG6TMCnCtBCC6TGGCCintGCC6CCI»86nnsyiim6CCGGC6T6TBC^^
Consensus CCTGdlTtffittniTcaiI « CCtfi6T6«fc iC a a iT B g j* rc 6 C tS ttS m 6 l^ ^
261 2 7 0 2 8 0 2 9 6 3 ( 0 310 3 2 0 3 3 ( 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 3 9 0
I ------ ■------ 1
nit TKCTTT6T66C66M
CT6BM
66TM
niS6CM
CCSM
6T6688ETCCC86rtllTTS6TSGICW
:n:CSE661CBCC8nCTIiCCTTTICHTCglBITCCCC66C6TC8SClTTH6C68TCm6Hfl
Hdh889El TKCTn(TttCEEKrEliG61Dni(X6CllIXGKT6aKTCtC66rnnT6EC6ETCKTCCE&G6rDIX8TTCTGtnTTICTETCHC8GITQIi6GC6TCfl6CFTTfl6C6flICfKGR8
T8CnTT(T6GCS6R6CT6MG6TsMlC86CKCEKET66flKTCCC66T cflTT66c8GICICTCC66G6rClCC8TTn6CCTTT8CT6iICaDI6inXCc66CST[f)6CIT T86C68TC866flH
391 400 <10 420 430 440 450 468 470 477
I----- 1----1
n il iTTHXa g CTSaCTIIBIII^lWnaM(CgXS6TIIC ta»>6rCSTCG4tf«C£fl(«(a:igafiCGgCSSKTCfiBCGIiXTT6Ta3nB
MM09E1 6T66CaaTCT^T(ifi*6CflTl[l(«fiCfiCC66TflCCfi«G[riTtifi(KG(^6G&C&6C£aT[5GCaKn6Ta3IT6
Consensus a |60^TGOIW^nCMVK6CCt6T8CCaU£TC6TGGmEtG6IIRGCG6GCfiGCGGGTI^S8CCTT(TC£llT6
258
Figure B.40 Alignment o f Pgi from K. pneumoniae AES809 sequence
1 1 * 20
I----- 1------1 - 30 40 50 GO 70 80 90 100
, — 110 -1 ,2 0— 1 3 0
|
P«i GH I
T CaK CKTB
W RTGn GMXSTM XGCtKGCf
iGTR GBCI
lCCHGR C
TGG CtC MntlT
CTff
iE GIEH
aXGGGC I
C D
K G ETC nC
BCG CtTTC T
RCCf
lGCTGliT
CD C ClKGG C KtlM
W
pgI8C9£l S^CCffffGGTWGTRTSrCSKtGTRKGGCtKGCt&TSGnCTHCDKKTGSCtXSfnCIITCT&GGGCGflGCC&GGCtCtfnCSiTCHGCfCGCtTTCTKXnGCTSRTCCflTCn^jCflCCIWW
i»Tmiih«TaaeTBTGTtamTW^nKcr^Tji3rnrrMfTtgiy^t^rTaa»riait«-ttiy«ff«iraiTn4riri«TTrTirra^gnTnfaniiy^fTHH0O
131 140 150 ISO 178 180 190 200 210 220 230 240 250 260
I ------------------- --------------- >------------- >----------------- m--------------- 1
P fiIGGTRCC&TGCGRTnC8TCGCTCr8GCTNTCHDX8PnCtCGCTCTCTG8D3CC8TCIEiMCTGCTCTtTRRCTTCTTCGCttflGKCGHGE(XCTGGCCTTTGCTIMITCCCGCG8HGTGGTTG8
P*I809E1 TSGTKtGTGCSRTTfCflrCGtCaWTRTDKCCKCMCCCGCTnCCfiRTCftlWCCGRKTGCTGTCaiCTKnCSClXflGiCCGRGGCCtTGGCnTCGGTIMTCCCGTGMTGGTGGfl
T6GTf(XBTSCfflTTTCfl!DiCctXG6CTHTCfl(XtfCfWCtXSCIcTCcGftcCftcCflTtaGflfi*CTfiCTSTCcflHCTTCTTC(KXCflGflCCG8G6tXCTGGCCTTcGSTftflflTCCCGcSflflGTGGTjGB
2S1 270 280 290 300 310 320 330 340 350 3G0 370 380 390
I............. — ■■t------- >■ — i ■ t------ 1.---- »-■ - i — I------- 1
P ji GCflGGHRTRTCSCG(ITC]KGETHnGiCIXSGC£KCtTGGKCKGTGETGtCGTTC8MTSrTCSRIIGETRBCC8CtCtflCTinnCaiICCTGCTGCGTt8GnTQXXCCGTTClKCCTCGGGGCG
wMOSEl QCHSaiPIlTCSCarTaiSSSTBHBGIfftJttCaiClX TiaiGCmGTBGTGCCSTTCagliillil II U H t i i m t U H t U UNI If TIIIII Ttf IM 11f Will 11IH f 11 III Tl IWiI | rrH4iliffi
GC8GG8PTRTCGC£ATC8GGEr8nnGAcCtSGC£8CtXTGGMXnCSTGGTGCCSTTC8RRGTGTTCE8RGGc(HXGCCCGflCTRfCTCc8TtXTGCTGCGTGHGiiiTCRClXtGTTCHGCCTCGGGSCG
391 400 410 420 43S32
I-----------------------------------------------------------------------
P ji CTGflrTGCCCTGT8CGHGC8CIWHWTCTTaKtOIGGtCGCG
PtISOSEl CTSHTT6CTCT6TBCSMiCKWTCTTfKC[MM[E[t
n»nGtcCTtTBcagaowBTcnaceiM«csa
Figure B.41 Alignment of GapA from K. pneumoniae AES809 sequence

10 20 50 70 100 110 120 130

—I
e*4 MXTBKT&anttfKTTGETETTUUn&TTUTUMCMXGGTIITCTTCCTUCCMCBWRCCSCTC6TMMCnCRTCICCGCTGSCOSflRlimeTanCTGICTGeCCanxME
wABOXl HXTGRflGTGGGMXRRETTGETEnGMXnCT7Gn0ilH3K£t&TinCTTCCTGinxaCGHnCCGCTCETMEM]nCKCGCTGGCSCGMMRGTCSTTCTGKTffiCCtSTCtMIG
Consensus WgCT6HflCT660flCSa86TT6£TETTS8nntTT6[TSRBtiCimfiSTinCTTmMCim M «aâffl(>naTaCIKrG6CSCaWWa6TaTTrTB«:TSSCtaTaaW
131
140 150 ISO 170 180 130 200 ao 220 230 240 250
I—
f* i OflCflCTCCBflTEnttTTCBOi&DXTHBCTTCSflCtCTTflCSCSDfiCOfiSRCSrCBTTTCDWCSCTTCCISCffDiCOWCTGCCTGGCKtSCTGSCTIBBGTTHTCflfiCfiflCMCTTCfiGfflT
npMMEi iQHCflCKcsflTEnanttTsggiiCTTCtaaKnacEasficattMaiTcsiiiUMiiaim6cictJcaKTSKTgc«xgT6B:rBaa6Ta>TtMiiMKTTutTiT
Consensus n)raxCCGRT61TCSTTCGc£GCSCTIKTTCGK4CTTICGCaGGCaBCilCGTTTn]|IK4T(nGCinKCnCT(itCTG&UCIXCTGGCT]WKTclTQMCSKIiCriCGET8T
261 270 280 290 300 310 320 330 340 350 3(0 370 380 390
I-------1-
prf aTT6flOe6a:T6«TMliaCCSTtaCSCT>CaiXSnKTOIMflMC£tnHTGga^TCTaa8««TBGCfiCSfiCfigC6CS6C6a6CTCttO(lCBTa)TaXfiTCt:TtTH:CS6CSC
japfl809El O TTSW 66aTMTMU3CCCTaJCfiCaHCUC£SaiCnjGWimtTTMTGttCC8TtTOClW ^rBGtfiCfi6C5Ktfirog:gH6Cra»«COTCHTOI6Ta:n:THCCSg6C
iHaHM njTTGfl(S(^TGflTBflCDKIfiT(IICEC4fCC8CCfitTflCTCflGflflflfCKTTSflIG6CCOiTCTC(C(llflGflCT8GC6CC6C6GCCCCCO:BCflGCICBGflfiraTCflrClX6TCCTtTtlCCSQ;s;
391 400 410 420 430 440 450
I- •I
e*4 SaiW60KTKET8ÎCT6CaK»TgW
miW
«TSflCCfiET8TEEC5TTC
Consensus 8CTimGQKTK6TMKTRCTKQiGRKTGnCfiGQVMCTGKCGSTRTBGCfiTTC
259
Figure B.42 Alignment ofPhoE from K. pneumoniae AES809 sequence
10 » 80 100 111 120 130

—I
PhoE GTCGK(ICCTCGTreflGCTflFTGRTTTCGGCS£CRGC£aCTTCGCCSTCflGCfiCRGCCTflCICUBCTCCSICCGTIICCmCGATCllGf)IICCTGCTSfiCCCGC6GCCflfififiTTCSi)aflBCSEIMGCC'rGS&
rhaEMSi sTtfflocnttnH«TircreTT(ma3iaKnc8amigreraTtiHiisCTgtttifiTitai»«TaB»rc
Comma* Enmcnt8TlllHTii«cniMStaBC£KnaiOTC8BCK^TiaCCKIHSICC5iraHEaiaS«CCTBn«raCKCaBtSIEaiBC6B»SCOi«
131 140 150 160 170 180 190 200 ao 220 230 240 250 260
I— —I
PhoE EiHi6iHTmTflTHK6mfloinBicnmaiOTaiETfcicrGfiflreaKB«anffliciiEmcflHms6cmK!Mfla(HH»itnTs»«CKT6ascflGT(iiai
PhoE809El COfCCtGCCTEftlflTftTB#COCIMflOTCrintT6SCCECtOTST(CTCr67WC1X{iTWKST8fl[C1XfiflTC8SCB6CtSCTnfiCI^(i66CfiC8(WCTTTSflH6C6GTG6t9Cltt'nCC9
Consensus CG)CCtGCCTUW
niT6KGCCM
CflRcflTCIM
lt{CGHCnrGTKTCTGflV)CIXGeiVGIIT6RCQIGRTC8GCSECtGCTn6CC]KfM
aGCGaaflCTTTG(nGCtGTIXCtC)KTlkDl
261 270 210 290 300 310 320 330 340 350 370 380 390
I— —I
PhoE GTTC£KTTCG£TCTGC£TCCSTrCCTCGGCTflTGTGCTETCGfWRGGGfMKSBTBTCGMGBG6rSGBSKTGfSlSflTCTGSTTMCTIDITCSSCSTGGGCCTGRCCTICTRCTTCflfC8lflflflCflTG
PhoE809El 6TTCEICrTCSGTCTGaT(X6TCCtrCUTRTGTGtT6TCEMIfiGGMCGRTHTCGRRGG6G1666EIGCEilS8TCTKTTflfCTCinTEK6T6GKCTGRCCTflCTRTTTCHCMfilUITG
SnC6Km»TCT6C$TCC6raCiCE[TRT6T6CTrafiM6Gfifil6SRTmC®fl666Gre6E6l6cfiaerCTaTimiOT<£flmBe6CaStttTKTIfcTiaitWiaT6
391 400 410 420

I— —I
Phrf wcacrrcsiGssnfiBtwiirfiCO«
PhoE809Q IICSCCTTUTURTTKlHMTCMCaK
Consensus

1 1 0 2 0 3 1 4 0 5 0 ( 0 7 1 80 90 100 110 120130

I ----- 1----- 1----- 1----- 1----- 1----- 1----- 1----- 1
tnoB ilCE rsarSDCKCCSPre nSttCCiSCmibtt'JfeCSOBCCTGraiSB*Ca^T6TT6»Ca5«IT^t^*CC**GTttTBni(WCCBCCB«tt«6C6a:uErSGIGiTa:
tnriSOXl (TeTGGCSCC^GflirTTGKXSUXC££Km£CMmTaêDKCaXTTETT»CnSf«XG0GCC£G^T16EC{n«TG:(XaKCSIXGmiEnGGCSCC&6TQGT6ITCC
h— iraiEimnxmntmsaxixsflmutciTcsTEdniEnsniMicMa^^
131 141 150 180 170 IN 190 200 30 220 230 240 250 260
|---- 1------- 4----- 1—-- 1----- 1—---1----- 1----- >-----1---- -I----- 1----- 1
tn i IT«HUXGnUTfVSIISnHXClVl(IITMIClIKBD^n6CCfiKnHMKrTB4KlfiC£GBSCK6IHSIBWX6ECKCfSRQXSXTrCKCCIIECSTTTGMMCRK]H
ti*6909El ffMICaS^THfKtEWCmWCTWlCaWtCiaBilKMHm
Consensus rW Di £ i C Tl » K i rS I K g l i8 K T l i( I C t o« K f I nf a ( E t6 i l ^
261 270 3>) 290 300 310 320 330 340 3» 360 370 380 390
|— H------------------1--------------- 1
---------------1-----------------I---------------1----------------* - -H------------------1---------------1-----------------1--------------1
tnoB THCfiSCiiCCSGEBCETfKISC^fKSOICCTCiflCflGCRfiCSBCrfWCHlfCCGTnC TSH COVtiSSCCC SCC

i&jCjITtflffiC&UTTC KCXST(XT1TCCflGCSCSCSCTCfftGCljCTSE
ti*69uxi iKscKiizsaaaiBzcaizi&TcaiXfftKs^
Consensus TKMgliBHraCi6ngdH3IITCdCiGOEBCc«CCOPM^
391 400 410414

I --------- 1- 1
tnoB HTSnGETKSTflCSSETMi
UmfiSOSEl fTT6*GfiWCaiKfiS6TBW
Consensus H T H K b C K I a C f i S l W
260
Figure B.44 Alignment o f infB from K. pneumoniae AES809 sequence
1 10 20 38 40 56 60 76 80 90 100 116 120 130

I 1-------- h--------1-------- 1-------- h— i------- 1--------1
infB GGC8raTaCriTCne&flTfimGîaC6CtGCSTTTBa:T(XflTG(£l%T(£TeGC&CGCflGGC£RCGGflTRTDiTe6TTCTffiTffiTCGCGGa^GR(mGTGflTGCtXCRGflCTflTC£
in fl& O S l fiGCflTfiflT(mTTaT6eiTfCra^GGCaC8(XfiCETTTimraiTBC8TGCTCST6eTeCGCl«GCSfi(^fiTRTCGTffiTTCT66T(iGTGGCfflCaQflCGflC6GCBTGflTSaB3«ittTflTC£
Constnstt SG»TSflT[mTTaTGGBrfCaXSGGCCfl(msaTnim(XflTBCGTeCTOTB&cfiaOSfiafCfifflTflrar8STTCTS6TSGTS6C£6CTSKfflCfi6CSTGflTBCCfiCmiflCTflTC6
131 ID 150 160 170 180 199 200 210 220 230 246 250 260
| H * •-------- 1--------1--------1--------I------- <-------- 1-------- 1--------1
infB fi8GC7flT(X}fiC«OTflflffttSGa^BG6T(CaXTffiT(€TSSCl^T6flfiCaf«aT(^Tf«GCa36aflGa^TCIOTrCSCET6flflQ«CSflfCTt7CIX*TfCGSDnCCT6CaiSaflGfi6re
MB809E1 TRT(XflKfiC6(Xf¥786C6GDjCaG61iC(^TGGTBGTTGC66TSfH3fiGflTf^TflBKCTBf¥BCt^T(X8GfCOTSTGaHGRftCS9CTETCCCBGTfCB6C8TCrTGCCSGfWGfST6
Consensus M im iIflG (H fita ll# 6 a ffS C IK T a lIttT 6 6 T a 6 T g ^
261 276 280 290 300 310 318

I 1 i — ------ 1-----1
in f l SGGCSSCSfiSRGIXHffrTCSriXflCfiTTICCSCBflRfiGCJGETflCCSGCfiTCBSCSaC
infB»;«l ffi6CSSCg36fffiCCfl6TTC£TCOC6TCT!XfiCfiHW6CCfiETflCCfi6T8TCSHCGflC
Consensus 6GfiCGGCGflGKCDfiTTCSTlDC£TcTCCGCGflBflGCcttTRCCGGdlTCGKGflC

1 10 20 30 40 50 (0 74 80 90 100 110 120 130

I- -------------- ------- - —t —«■ — i t--------- +---»------ 1
RpoB fCrXKTf€MTC6C£TflTSrC£fiRTCGfift«&CrTGflfiGGTaGfiftCfiTC(iGTCTGflTTf»CrCCCTGTCCGTSTfCSC6Cfi8a[XfKCGfifirnTGGCTTCCTTGflG«£CC6TfiTC6TflF«6TaC£ll
HMD fCTnJCT(CGGTCfiDjT(IT5TtlSHTCfiHflOCGCCTGfiflGGTCM flflCflT(^T(rrG4nO>CTtXCTGlTCfiTHTfCfiCSCnGliXflfCfiHflnrrGGCTTCCTTGflGflCBCCBTflTCGTflfifl6T6flCCS
Consensus fCcCBCTfCB6r(^CEIflT6TI)CaflTCHWflC6CCTGRH66T(XEHHCTTI®T(rrfiHTcRBCnXCTBlXrfiTaTHraCSC8HCCflHCfiHfiTfiTS8CrrCCrTSW7CfiCC6TflTC6TWWjT6fiCCa
131 140 150 160 170 180 190 290 210 220 230 240 250 260
| 1 i —♦ -h------ —---► --- *------ *------ 1
M ftCSGTGT6GTT^TSfiCSJ«HnDCrfCCTGTCTSCTflTCGf«GrtRGGCJW TfCenaTCSCTC*GC6fWCTCCmCCTGSHrSflfiflflC6Ga:flCTTCfiTnGfW
GaiCTGGrntCCTSCCBTRGC«fl
M 8 0 9 E 1 RCfiETSTGGITiCCGRCGRHHTTClCTRCCTGTCTGCTHTCWRGHRGGCRflCTflCErTRTCGCTCHBGCSfMCrCCRRCCTGSIT&HRRHCSBCCRCTTCETRGflflGRTCTGErGKCTGCCGTflGCMI
Consensus i^GGT6TKTT(CcGflCGflM nM T(CCTfiTnGnflTC(iflfiGfW
iG(M
T(CCnHllGCTC8fifiC8M
CTCCflflCCTGMTHHiflCGGCCflCTTCGTflGflfl01TCTGfiTgflCCT6(XGT8GCfffl
261 270 290 290 300 310 320 330 340 350 360 370 380 390
I----- 1----- f- ■ '------ 1------ 1------ 1—--- 1------ 1
R pri 86GCfiflfiT(I8fiC[T6rrClUI8C£flQ^!T68CT(OTS6flCfiT8TlIBCIIfi8C1®IS6TfnKSTCB6TGC6TlXCTGHTIIC6TTlITGGBflC8CfiflTGflCGCniCCSTGC8T7fflTGG6I
Rpo8909Q f^68fiT(X86CTTGrrQ^ffS9CCft66TT6flCTIOTG68C6TfrrCC8CCC86C8G6T(j6TflTCC6TC66TGCfiT(XCT6flTCCC6TTCCT6Sfi8CflCG0TSfiC8CCflflCCJTSC!ITTlifiTW
6T
WSC^Tn3»ffTTSTTC«GCC6CS(Ca»ttTTOICT(OTSB»CSTfriXaiCro»6ClS6TttTHTrC6TCSGTSC6T[XCTSflTraXTTaTSGfiBCBCGflIGBC6Ca«CC6TGCflnSfiTGGCT
391 400 410 420 430 440 450 460 470 480 490 501
I ------ -•-----— h-------♦--- »------- h------------------ >|
-
RpoB SCaHBCHTSCWCSrCflGBCBSTTCXSHCTCTSCBCaCTSBIHNGCtBCTSGTISEISCCSGTflTBGHflCSTSCTSTTiaXSTTSHCTCCBBTSnaCTHXfiTSGCTHHfi
RpuWSEl GCfiflllCfiTGCIIK6TC8GSX£TTCCatTCTGC6CfiCTGflTflfiGCCECTGGTTG6TKCfiGTflTG6MC6TGCTGTB(iCt6TCGaCICCGGCGrr8CTGCrGTTGCTflfl6
Cmcmb 6CSnflCHTBCflHCBTCflGBCcfiTr(XfiHCTCTGCBCfiCTe»THHG(XljCTS6nS6T(CCBBTgTBGH8C6IGCTfiTaaCCSTcfiHCT(IB6cfiTTHCTGCCfiTfGCT(IHa
261
Figure B.46 Alignment ofPgi from AT. pneumoniae AES808 sequence
1 1 0 2 0 3 0 40 50 60 70 88 90 100 116 1 2 0 1 3 0
I — *------ 1
Pgi 68GTniHraTiKIin6TTGRDXTMCSGIXilHSGTIIGRnflCIXKT6(CaillHTQ)TCT6GGETG8GQ£GGCflQnC6GTQIXfCHXTTCTRCC8GCT6RTCCfCCWG6aCOMVn
P|iSanpleW a Sf#TCCflRT6CTMGTflTGTCfiB(IST(ICSGCOCS(XSTB^8CCIB8n6GCOWffiTCI6GG6CafiCtffiOIXIM®TCflGCICfiCBTTCTlCOBCTQnC01TCH6G6CHXIfflflfi
Consensus L«TCCf»k^TSR6mTGIc5»CCGTf«SGCCICia^TaGnCTBCaOKTSGCCCaflTcflKT&GGGcGfl6CCG66CfCCHK&6TaiGCfCQ^TTCTlCCfiGCTGflTCCfk£flGG&f3flCCf¥lRfl
131 140 150 160 176 180 190 200 BO 220 230 240 250 260
| 1 ►
— 1 1 1
Pei TG6TBCC£T6CfiflTTTC8TreCT(XGttTflTacn30fltt£fiCTSTrrG8CCflCDITDfeflWKT6CTGTn(HrncnCfi(XC(ttfl(IfiflGfiCn;TGfiCCTnG6TlfliTC£££Cfiflfi6TGfiTT6fl
F e iS n p la lM T6TlISTSCfim 7raT(m a66CTrU ClX fl(m iX BCTCTCnifiTam j^C CH I«CT6tTEnX *CTTCTTCSCII*ttC86fiCO :TB6a;nCfi6T(W TaiEraK TB6T6»
Consensus T6CTRCC6TH»TTTCRTCSCe(ISGCTBTa*IO*mCCBCTcT(xafcaii£»ICaG^
2S1 270 280 290 300 310 320 330 340 350 360 370 380 390
I--------- h --------- t----------- - — t ■ 'I ----------- 1 ----------- 1
Pgi GQfi&MT6TC6C£8nSGKTRRHGflCIXSGCGRQXTGS8GQnTGGTGaXTTCMWlCnC£flKETiCIXDX6MinniIHTIIT8:iGCGTtnG8TC)CDX6TTt8C(XItGGG6CG
P |iS a v le M 9 6L8B6MTITCSCMTC^T8flB6W ngC fiK C C T tfltfin i6raC<^ nCMW TCnra88S6CBCtSCCCfifl[nWCTCTHI(I16CT6C6reflttTCWXCCSTTDCtIKfi66g6
Consensus SaC«OTTtSCM Ta«6TBf«GfcCtgCM CCCTim ilST66TCCC8T^^
391 400 410 420 43032

I H ----------- H|
Pgi CTGHTTGCCCTGTBCGRGCaQWfWTCTTCMXCfiGGGCGCG
P e iS a v le O M CTBHTTBCTCTGTBCfiHQCHCflfiHHICTTOCCCflSGGCfiCE
Consensus CTE*TT6MT6T*5E3iaE«TCTiaCCC8B66C6C6

1 1* 21 30 • 50 SO 71 80 91 1M 110 120 130

I— — l 1 . f— t I---------- «------ I
t i« s 8T S 6T ssestts6trsrm 6«cts[ritiS G C S S cst8gtT n gias6trrsT iS T T B w cct BwccTs i « c g « c c « B « is s w t c T s ascc g c g w w sflcscts 6TKTafTtt
umM osb »T6ET»csnm tsaTm sKtTEcttctsQcsstscM cttxrcfTsc«Gn:tiTTsin8accraiixGsaBccscM cns«nciictSGaoccsnnM GKScscts6TSETG(iT<;c
Cw m mS6T6aCSCtSSCtGnrnaKtISCtcCtSaCSS(SOWt^CSIGcffitCtSmnHmc«MCt|SRBCCSGMtjtWGTrfIcCC|eiWCCSCI»W8ESCGCtSGTGGTSnTCt
u i # » i » » i » » » t a t a i » » » 260
I f . » » ■- - « I - I --------- 1 ■> ■ -I
M B7FMCCSli^ T B H tC C «K C aM K XTR8K afla6(XTaK a£fi»K M 6m m K U »K <^G H yK 1SM K IK W XK aranX& 6tCT(m STTT 6W W M Cfl8
m m w B t iiM C M ^ T iiig csaagcnHSCCTM K tM K m ig r r t a a wHBK s n tM CK c g iw sc sg sc sT rs HiK sg c s c a iisctscsreciScnutcsT ntw w iK M O H
Em aa) Sl»M tIJBK tl8H g a W g U SISKCniK t cgS6CU9Mgg « a« lll^ t6hC SSU H »C SB SW c W ^ Q a :ig 8 B IS g gS rtC S B C clU C n n saHK flroil
261 flQ 288 290 300 310 380 331 3# 358 360 370 3W 390
I— ... ----------- 1 , < -« --------- 1--------- 1------- —t -------- t -------- »--------- I
tm B i K m g n m m e K m t n m s c K X T i m K c m c m m M a H x s r K i a i u K i x s e c c a a t t i a T a K a c m a w sT a m t t m u a a c n K s c s n e t t c
tm w o s s TiriyTaTiiB^ t f irrtfO T OK fiiT r n w i s a s f M r a i i ^ t n m m c T t a i a B s ts t tits c tH a M T tiis m c fin im tH itr m c g s ttc sa c T a itg t CTsrsf
C a tta im Tiru^^SC6cKaGCaCtdtt(3CrTCc«tfCWmd«KaXt6rdCT6CeCQmXGaXStSGGCSflm6CCSCGTICIGCtGaCmttaGCSCStS:TaiG6CSneC6C
391 400 <tt* <H

I -■ I— I
tn rf SnGSMSTmSTKSSTSOK
tnaMOSB CTiM MM XSIKttGTMK
C om m as SlIGSHGUCGSItCSESTSMt
262
Figure B.48 Alignment o f PhoE from A', pneumoniae AES808 sequence
1
I—1 * ------•
20
------30 40 50
>------GO 70 M
1------90 100 110
1------1 2 0 1 3 0 1
PfcaE GTCSSnC£TCSTTMffTIITGirnCK£SGaBCSKTTCBGCETOSCGCKKTKKtKCTCCSKCtTKCMCSRTCMKtTGCnKCCGCSGCCWGEntSMMGCGGMGCCTGGG
U N E S o p la M I Gn&SKnnTTIKCTfTGKTTCGBCGGCKCKTTCKCSTOSCSGSGCCTKKCMCTCCGKCSTKCMKSnCMKtTGCTGGCTCSCSEIOBGGTTCGMKCSGMGCCTGGG
GTc££cflCtTCGnRniXTfnGncTTCGGCGGCflGCGICrTCGIXGTCnGCCt«GCCTHCHCCnGCTCQjflCCGTnCDnCGnTCHGHRCtTGCTGGCcCGCGGcCnGGGTTC£nHnGCGGfinGtXTIX&
131 140 150 ISO 170 190 190 200 210 220 230 240 250 260
I-------1--------1-------->------- 1-------- 1--------1--------1
ftf MhW csictgn:TG0iigniTGgKU«KJ^TCTgTCTSGcymTEncTtTGiiMttcGrM
6»TyctaMTni<irujHJTTTwrnnrnnnM
'GfiiiinnrTTT‘:anc^ TCO~sracTgTca
CoroenaB C6ICCSCCCTSIWingTGWJIXaBOWciTCT>d^B6Cfi>IIX8IGTHCTCTt8 IICrCfcJBGBTG8CtCCGHTCllGCSGC6GnTT6CtaiC8BHGCGCWGHBCTTTGBBBCG6T6GC6CB6TBTCB
251270 280 290 300 310 320 330 340 350 360 370 380 390
| 1 1 1 — -» . » 1 1 1
PM 6TTCGHCTTCS6TCTSC6TCC6TClXTCfi6CTHT6T6CTGTC6BB8666W8658T8Tt68B66G6T666GWiTGBB6BTCTS6TTH8CT8C8TCS8CST666CCT6flCCHlCTICTTC8BClWHB8C8T6
PhoESMfleWH 6T1CS8CTTT66TCT5C6TIXGTIXCTCla£CTI^676nGTCGRHH6GGRflGGnT1)TC6RRGGGGTGGGGB6CGMGHTCTGGTTIICTnCI)TT6fHXTGGGCCTGHCCTRCTICTTC8RC(n(MnD)T6
TTCSHCTTc66Tn5C6TCC67CttTCC6CTIITET6CT6TC688868EflBGMTIITCaW6666TGCGG8Cc6miG8TCTG6TTflBCT8C8TcCHCCTCGGCCr68CtTICTICTTCH8CBBaiWC8T6
391
I-----4001------1
410
-----4201
P M MCfiXTTCSTGGfrTRCMMITCMCCflE
PhoESaipleftMe MK6ttTTCBI6S8TT8CMMTOMCnK
ConseRM* RnCGCCTTCGTGGHTnCMWNTClHX8G
Figure B.49 Alignment of infB from K. pneumoniae AES808 sequence

1 1 1 20 36 40 50 60 70 80 90 100 11(1 121 130

I > —«-------------- t----------- 1 ■■ i ----------- 1------------ 1---------- 1— ---■ H 1----------- 1
i o f l raTG
fiT0CCn(XTS6flTnCaXffiGCaCfiCl^mfCCTCraTGCfiTBCTCBTGGCaCSCIfi6Cfifl(^THrareGTTCTS6T6GT9GCGO6flCEflCSGCETaiT6CCaC8fflCTRTtfi
infBSanpldXM SGCflTSflTCflCCn(XTBfifiTH(mSG6Cat8X6CSnTfCCT(XflTGCETSCTCGTG6TS(^fl6GCfiflCGGflTRTCSTG6nCTG6TGGT(»CGeCftGfl(mSGCGT6fiTe(XSCflG«:TfiTtS
(m m ^TaiHfTTTriTCmrrrîfgfOTTTTgrTrmfffiTimTttEggMrM^
131 140 151 ISO 170 180 190 200 210 220 230 240 251 260
I------ ,------ 1------ 1-------1------ 1------ 1------ 1------ 1------ 1------1------ 1------ 1----- 1
in f ! K C m iX I f f lC O m C f f l^ T I K t M f iT lf iT G G C t t T lililC ll^
jjrflSaplaW
Consents I M G a ilT I I I £ # X b a ilK ! E S lM a Q M ^
261 271 280 290 300 HI 318

I -h h 1------ 1
in f ! SGG(m£fl6ffia^nraTCCflCCmCl^CfiftfiflGCfiGGTf«mflT[SCSfC
MKapleM SGSfmfiflGfl^flGTraTraOlClCt^CfiflfifiBaiffiTfCCGCTHTCfiftCfifiC
C onsenss S G G O X C a fifiX i^
263
1 II 21 31 40 51 (I 71 80 90 100 110 120 130

I----- 1- - - - - - - - - - 1- -------- 1— 1- - - - - - - - - - - - - - - i---- 1
t p l ftCCCfCTfCS8TCfiCfiTflT6TCC®BTC6flflflCSCCTSflflS6T(XfiftflCfiTCB5TCTG#lTTR(CICCrTGTCCSTBTflC6C6CflGflCCHflCfiflflTflT&GCTTCCTT&flSfiCQCC£TflTCfiTflflflGTSICCfl
M h jfc — RnC(CT(C66Tt^M6TCCJI(iTCfiflfl(ICfiCCTSflflG6TCC6ftttflIC56TCI6flItflCTCCCTGTCCfiTflT(tGC60fc<CCfWC6flflTBI&8CTTCCTTGfl(iflCGCC£TflTC6ISftflGTSftCCJ
c « m f f f - ^ Tr a r m m f f T r r ^ T r a n r ir r iH ^ r y O T r r g ir T M ii T T nrT n im iT inrnrcririirriMrMinnTnnrTTrrrnrnnnnj i h i i i m u w i a r i
131 140 150 160 170 180 190 200 210 220 230 240 250 260
I 1 ■■■■-i---- —«----- ------- ------- ------- -------------- ►
—--- 1
Rpri flCG6T6TS6TTflCT5ACfiAAflnOCTICtT6TtT6CTfiTCSI)fiAR66CflCTlC6TTRTCS[TD66CSIICTCQWC£T66flT6flflflltt66CCltTTC£TBfiBfl6flTCT66TTflC[T6CCiTl6Cflfl
R p o lS a p lfM i HMTilttlliCtittMlintiCTIClTSTCTSCTHrCWIiyHiMIWlTKIlllllillOMUilLICCiWXTIOTIIIUCMKKTTKTWIIIMTCTitTMLlMtiliyjl
Consens* (CffllSISBTTlIcSKSflRfiTIOCTlITBrcTttlRTlBBGflflGHMnoniTCfflDfiECGfflncailllTtfflTGfllMCGGCaCllCEIfBfllliRrClfiBTelllTGCCBTRGCflfl
281 270 280 290 300 310 320 330 34) 350 360 370 380 390
I -------- 1-------- - >— -I------- 1................ 1----1
Rpal a 6 g ^ T C a gTTSTCIgCaCMtri>tSnBCTOTSai^CtiXa>6Cl»Tli£THTCC8TCaT6C6TCtXTMTClXSTTn:TSBIfOCSRTMC&CCM»XCT6aTTMT6fi£T
H j u K f k W M g a r o i l gTTBTTa i a a i M C Ig TTttCTiaT B M C tT O Itie tC M a ^ T IIT O S T M T S ^ ^
Corewat (UX^TIIRGCTlBTTnfiXGCGRXRGGnGHCTlOTtHCEMIXRflCnGCRGGTGGTirilGTDXTSXTCIITGRTniGnCXTGGflRCRCGRTGRC&XlllISTGtSTTGflTGGGT
391 400 410 420 430 440 450 4S0 470 480 490 501
BC6rfOI5Cf»CoTln6blR[ n.lan[TLi BLGLSCTGfiTSRhCChLTSbTTKTHCl^THTGeWK:CTBCTfiTTGLL5TTSnCTC T CTSCCfiTSGCTnflH

RpoBS«ple8088 KSflflCflTSCfldCSTCflGfiCCGTTCCfiftCTCTGCGCSCTGRTRWCCGCTGETTGGTRCC&ETItTGSHC6T6CT6TRGCCSTC&RCTCC6SC6TTRCTGCKTSGCTMG
SCSRRCRTBClltETaBttefiTTCtSETCl GCEC&CTSATHRSXfiCTGGTTGGTKXSGTflTGSflRCETSCTETaBCCBTcfiflCTCCGGcurmCTGCcfirSBCTHRa
Figure B.51 Alignment of mdh from K. pneumoniae AES808 sequence

111 W 30 40 54 SO 71 M 90 100 110 121 131

I■ . ■ t-----*------ 1
■* B£MMi8[»iaiOTiaggHCTsc^Ti»cnmiTOTgniiîintMitiwi8îingiMHa»cCTareaiajiHiTTKtMM(xiictgaa
n*sapkiNi BtgTBTSTitTgiannagwarotogTiiBPXBunwgnamitTniniTMorocMroittiiiB^ TaTiicwiiiiimKccwKnKixaig
Cm m ec^TGT^TKTammKttficnjBcoaTnEaxsacmsEHTanasKcronrmgTGMTSCjeETfncsifflMMcaaTEKcnnaBxmKciBXCKKt
1I31 140t 15i0 1■(0 170 1M 1i #----- -t-----
*.....................
200 210 22t-----■
0 23---- 0 240— 2--------51 2»1
n* CCT6OTFCffiCOrriTDCCKIX£0GM IKaCC(TtNITQCiaCtEflKIKTHRflR4RGCI3BC£1EIK&flTM
MnCM
KltTTDSCSrTICQCGCTG(fI1ITCtTCCSrTCOI
iASa>ieMB ccTffRii»DiniTTHcam(^namKcnKtcfiK!ixcEfneiEr8HiM«cs6CBTsiKSRmMn(^Tsmo£ixiSfmcsaawinaicimm
Cnesse CtTanTCSgBTmdlI«rC8(TBfc«IllgfTgtc»raaHSM«rfIBW W«CCC(^^
I > ■
.—........*-------♦------ 1 —*■ ■■■■ i — t ■■■ ♦ ■■ «■■■■ I
rt TlKTTTiT6SCSaKW WKIIBICttO)KC(*STS6*SI!lCSSTC8nSBIBGTCIinCtB86IOCCBnnfiCnTTOCISniDSHTCtiIGE(IfCISCTTTiSCWIC16Sfli
11*7C
amIm
»w TirrTTTBTW,BariB*«iTOTar«rfMMTff« TrrrmTnTTBiritTrirTTTtt«T
m TTr^'r^Jryg^ll'iri^^ta^â^ M^rrrBnrinBertagrTrfEM
ri»TinnnnH
CiriiiT ntrrm
-fTnnarrtirjfiaB
nT;TnîTrrfgantrTniKrttTriiMi
n M 410 420 430 440 450 480 470 477
I i .. »■■'■■■«'■ ■>■ «—■—* I
r* irTSQ
SCCTCK.TM
K0RnDSIKSQSlCaM
(T(STBSRREC£il)HBC85BCSSCfifiETCSSBICtnElCSfnG
nf&aapleMB iTKaMCTGKT0K£ainCMMCBIW KC{S(mET66MKCEIMCC£8^GB61!XSCaCCneiCanB
caww «T|6aMri:TsyTiiHartnpawgnciKcaâtE6fiH6aw»rsgfiifirgsTcsscBictntTgi>TE
264
Figure B.52 Full sequence o f AES81 integron
cttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggctt
gttatgactgtttttttggggtacagtctatgcctcgggcatccaagcag
caagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacga
tgttacgcagcagggcagtcgccctaaaacaaagttaggccgcatggaca
caacgcaggtcacattgatacacaaaattctagctgcggcagatgagcga
aatctgccgctctggatcggtgggggctgggcgatcgatgcacggctagg
gcgtgtaacacgcaagcacgatgatattgatctgacgtttcccggcgaga
ggcgcggcgagctcgaggcaatagttgaaatgctcggcgggcgcgtcatg
gaggagttggactatggattcttagcggagatcggggatgagttacttga
ctgcgaacctgcttggtgggcagacgaagcgtatgaaatcgcggaggctc
cgcagggctcgtgcccagaggcggctgagggcgtcatcgccgggcggcca
gtccgttgtaacagctgggaggcgatcatctgggattacttttactatgc
cg a t g a a g t a c c a c c a g t g g a c t g g c c t a c a a a g c a c a t a g a g t c c t a c a
ggctcgcatgcacctcactcggggcggaaaaggttgaggtcttgcgtgcc
gctttcaggtcgcgatatgcggcctaacaattcgtccaagccgacgccgc
ttcgcggcgcggcttaactcaggtgttatgccgcactcacccccatggag
ttttgatgttcaaacttttgagtaagttattggtctatttgaccgcgtct
atcatggctattgcgagtccgctcgctttttccgtagattctagcggtga
gtatccgacagtcagcgaaattccggtcggggaggtccggctttaccaga
ttgccgatggtgtttggtcgcatatcgcaacgcagtcgtttgatggcgca
gtctacccgtccaatggtctcattgtccgtgatggtgatgagttgctttt
gattgatacagcgtggggtgcgaaaaacacagcggcacttctcgcggaga
ttgagaagcaaattggacttcctgtaacgcgtgcagtctccacgcacttt
catgacgaccgcgtcggcggcgttgatgtccttcgggcggctggggtggc
aacgtacgcatcaccgtcgacacgccggctagccgaggtagaggggaacg
agattcccacgcactctctagaaggactctcatcgagcggggacgcagtg
cgcttcggtccagtagaactcttctatcctggtgctgcgcattcgaccga
caacttagttgtgtacgtcccgtctgcgagtgtgctctatggtggttgtg
cgatttatgagttgtcacgcacgtctgcggggaacgtggccgatgccgat
c t g g c t g a a t g g c c c a c c t c c a t t g a g cg g a t t c a a c a a c a c t a c c c g g a
agcacagttcgtcattccggggcacggcctgccgggcggtctagacttgc
tcaagcacacaacgaaatgttgtaaaagcgcacacaaatcgctcagtcgt
tg a g ta g caggcag a tg cggc a t a a c a t g aag t t g cagccgaccat cact
ccgctgcgctccgttctggcggctgaacttcggcgttaacctctgaggaa
gaattgtgaaactatcactaatggtagctatatcgaagaatggagttatc
gggaatggccctgatattccatggagtgccaaaggtgaacagctcctgtt
taaagctattacctataaccaatggctgttggttggacgcaagacttttg
aatcaatgggagcattacccaaccgaaagtatgcggtcgtaacacgttca
agttttacatctgacaatgagaacgtattgatctttccatcaattaaaga
tgctttaaccaacctaaagaaaataacggatcatgtcattgtttcaggtg
gtggggagatatacaaaagcctgatcgatcaagtagatacactacatata
tctacaatagacatcgagccggaaggtgatgtttactttcctgaaatccc
cagcaattttaggccagtttttacccaagacttcgcctctaacataaatt
atagttaccaaatctggcaaaagggttaacaagtggcagcaacggattcg
caaacctgtcacgccttttgataccaaagagccgcgccaggtttgcgatc
cgctgtgccaggcgttaggcagcacagttagcgaccatttcaatgtccgc
gagcaccccccccataactcttcgcctcatgaccgagcgcgacctgccga
tgctccatgactggctcaaccggccgcacatcgttgagtggtggggtggt
gacgaagagcgaccgactcttgatgaagtgctggaacactacctgcccag
a g c g a tg g G g g a a g a g tG G g ta a G a G G g ta G a tG g c a a tg ctg g g G g a g g
aaccgatcggctatgctcagtcgtacgtcgcgctcggaagcggtgatggc
tggtgggaagatgaaactgatccaggagtgcgaggaatagaccagtctct
ggctgacccgacacagttgaacaaaggcctaggaacaaggcttgtccgcg
ctctcgttgaactactgttctcggacccaaccgtgacgaagattcagacc
gacccgactccgaacaaccatcgagccatacgctgctatgagaaggcagg
attcgtgcgggagaagatcatcaccacgcctgacgggccggcggtttaca
tg g ttc a .a a c a c g a c a a g c c ttc g a g a g a .a a .g c g c g g tg ttg c c ta a .tq c
ctaactcagcgttcaagccgacgccgcttcgcggcgcggcttaattcagg
265
c g tta g a tg c a c ta a g c a c a ta a ttg c tc a c a g c c a a a c ta tc a g g tc a a
g tc tg c tttta tta ttttta a g c g tg c a ta a ta a g c c c ta c a c a a a ttg g
g a g a ta ta tc a tg a a a g g c tg c g t
Figure B.53 Full sequence o f AES83 integron
g a a c c ttg a c c g a a c g c a g c g g tg g ta a c g g c g c a g tg g c g g ttttc a tg
g c ttg tta tg a c tg tttttttg g g g ta c a g tc ta tg c c tc g g g c a tc c a a
g c a g c a a g c g c g tta c g c c g tg g g tc g a tg tttg a tg tta tg g a g c a g c a
a c g a tg tta c g c a g c a g g g c a g tc g c c c ta a a a c a a a g tta g g c c g c a tg
g a c a c a a c g c a g g tc a c a ttg a ta c a c a a a a ttc ta g c tg c g g c a g a tg a
g c g a a a tc tg c c g c tc tg g a tc g g tg g g g g c tg g g c g a tc g a tg c a c g g c
ta g g g c g tg ta a c a c g c a a g c a c g a tg a ta ttg a tc tg a c g tttc c c g g c
g a g a g g c g c g g c g a g c tc g a g g c a a ta g ttg a a a tg c tc g g c g g g c g c g t
c :a tg g a g g a g ttg g a c ta tg g a ttc tta g c g g a g a tc g g g g a tg a g tta c
ttg a c tg c g a a c c tg c ttg g tg g g c a g a c g a a g c g ta tg a a a tc g c g g a g
g c tc c g c a g g g c tc g tg c c c a g a g g c g g c tg a g g g cg tc a tc g c c g g g c g
g c c a g tc c g ttg ta a c a g c tg g g a g g c g a tc a tc tg g g a tta c tttta c t
a tg c c g a tg a a g ta c ca c c a g tg g a c tg g c c ta c a a a g ca c a ta g a g tc c
ta c a g g c tc g c a tg c a c c tc a c tc g g g g g c g g a a a a g g ttg a g g tc ttg c
g tg c c g c tttc a g g tc g c g a ta tg c g g c c ta a c a a ttc g tc c a a g c c g a c
g c c g c ttc g c g g c g c g g c tta a c tc a g g tg tta a c c tc tg a g g a a g a a tt
g tg a a a c ta tc a c ta a tg g ta g c ta ta tc g a a g a a tg g a g tta tc g g g a a
tg g c c c tg a ta ttc c a tg g a g tg c c a a a g g tg a a c a g c tc c tg ttta a a g
c ta tta c c ta ta a c c a a tg g c tg ttg g ttg g a c g c a a g a c ttttg a a tc a
a tg g g a g c a tta c c c a a c c g a a a g ta tg c g g tc g ta a c a c g ttc a a g ttt
ta c a tc tg a c a a tg a g a a c g ta ttg a tc tttc c a tc a a tta a a g a tg c tt
ta a c c a a c c ta a a g a a a a ta a c g g a tc a tg tc a ttg tttc a g g tg g tg g g
g a g a ta ta c a a a a g c c tg a tc g a tc a a g ta g a ta c a c ta c a ta ta tc ta c
a a ta g a c a tc g a g c c g g a a g g tg a tg ttta c tttc c tg a a a tc c c c a g c a
a tttta g g c c a g ttttta c c c a a g a c ttc g c c tc ta a c a ta a a tta ta g t
ta c c a a a tc tg g ca a a a g g g tta a c a a g tg g c a g c a a c g g a ttc g ca a a c
c tg tc a c g c c ttttg ta c c a a a a a g c c g c g c c a g g tttg c g a tc c g c tg t
g cca g g c g tta g g ca g c a c a g a g c g a c c a tttc a tg tcc g c g a g c a c c c c
c c c c a ta a ctc ttc g c c tc a tg a cc g a g c g c g a c c tg c c g a tg ctc c a tg
a c tg g c tc a a c c g g c c g c a c a tc g ttg a g tg g tg g g g tg g tg a c g a a g a g
c g a c c g a c tc ttg a tg a a g tg c tg g a a c a c ta c c tg c c c a g a g c g a tg g c
g g a a g a g tc c g ta a c a cc g ta c a tc g c a a tg c tg g g c g a g g a a cc g a tc g
g c ta tg c tc a g tc g ta c g tc g c g c tc g g a a g c g g tg a tg g c tg g tg g g a a
g a tg a a a c tg a tc c a g g a g tg c g a g g a a ta g a c c a g tc tc tg g c tg a c c c
g a c a c a g ttg a a c a a a g g cc ta g g a a c a a g g c ttg tc c g c g ctc tc g ttg
a a c ta c tg ttc tc g g a c c c a a c c g tg a c g a a g a ttc a g a c c g a c c c g a c t
c c g a a c a a c ca tc g a g c c a ta c g c tg c ta tg a g a a g g ca g g a ttc g tg c g
g g a g a a g a tc a tc a c c a c g c c tg a c g g g c c g g c g g ttta c a tg g ttc a a a
c a c g a c a a g c cttc g a g a g a a a g c g c g g tg ttg c c ta a ca a c tc a ttc a a
g c c g a c g c c g c ttc g c g g c g c g g c tta a ttc a g g c g tta g a tg c a c ta a g
c a c a ta a ttg c tc a c a g c c a a a c ta tc a g g tc a a g tc tg c tttta tta tt
ttta a g c g tg c a ta a ta a g c c c ta c a c a a a tn g g g a g a ta ta tc a n g a a a
99
266
Figure B.54 Full sequence of AES 135 integron
c g ta g c tg ta a tg ca a g ta g c g ta tg c g c tc a c g c a a ctg g tc c a g a a c c
ttg a c c g a a c g c a g c g g tg g ta a c g g c g c a g tg g c g g ttttca tg g c ttg
tta tg a c tg tttttttg g g g ta c a g tc ta tg c c tc g g g c a tc c a a g c a g c
a a g c a g c a a g c g c g tta c g c c g tg g g tc g a tg tttg a tg tta tg g a g c a g
c a a c g a tg tta c g c a g c a g g g c a g tc g c c c ta a a a c a a a g tta a c c c g g g
a c c a a a a ttg tg a a a g ta tc a tta a tg g c tg c a a g a g c g a g a a a c g g a g t
g a tc g g ttg c g g tcc a c a c a ta c c c tg g tc cg c g a a a g g a g a g c a g c ta c
tc ttta a a g c c c tg a c g ta c a a c c a g tg g c ttttg g ttg g c c g c a a g a c g
ttc g a a tc a a tg g g g g c g c tc c c c a a ta g g a a a ta c g c g g tc g tta c tc g
c tc a g c c tg g a c g g c c a a ta a tg a c a a c g ta g ta g ta ttc c c g tc g a tc g
a a g a g g c c a tg g g c g g tcta g c ta a a c tc a c c g g tc a cg tta ta g tg tc t
g g tg g cg g g g a g a ttta c a g a g a a a c g ttg c c c a tg g c c tc ta c g c tc c a
tg ta tc g a c g a tc g a c a ttg a g c c a g a a g g g g a tg ttttc ttc c c g a a ta
ttc c c a a cttc ttc g a a g ttg tttttg a g ca a c a tttta g ttc a a a c a tt
a a c ta ttg c ta tc a a a tttg g a a a a a g g g tta a c a a a g c ta tg c a a ttg a
c g g c a a a a a a g c ttc g ttc g c c g c g c tc a c ta c g c ttttta c c g c a a ttg
a ta g c g g c g tta g a tg c a c ta a g c a c a ta a ttg c tc a c a g c c a a a c ta tc
a g g tc a a g tc tg ttttta tta ttttta a g c g tg c a ta a ta a g c c c ta c a c
a a a ttg g g a g a ta ta tc a tg a a a g g c tg g c tttttc ttg c ta tc tc a a ta
g ttg g c g a a g ta a tc g c a a c a ttc g c a tta a a a tc ta g c g a g g g c ttta c
ta a g c ttg c c c c ttc c g c c g c tg tc a ta a ttg g tta tg g c a tc g c a tttt
a ttttc tttc tc tg g ttc tg a a a tc c a tc c c tg tc g g tg ttg c tta tg c a
g tc tg g tc g g g a c tc g g c g tc g tc a ta a tta c a g c c a ttg c c tg g ttg c t
tc a tg g g c a a a a g c ttg a tg c g tg g g g c tttg ta g g ta tg g g g c tc a ta g
tta g tg g tg ta g ta g tttta a a c ttg c tttc c a a a g c a a g tg c c c a c ta a
ta a a c tc a g tc a tc ta a c a a g tc g ttg c a g c a c c g c tc c a g c a c ttc g tg
c c tg c g c tg g a c a g ttttta a g tc g c g g c ttta tg g ttttg c tg c g c a a a
a g ta ttc c a ta a a a tc a c a a c tta a a a a c tg c c g c tg a a c tc g g c g ttg a
a c g a c a g c tttc c c a a a a g c tc ta c g g c tg c tc tg g g tc g a c a c c g g ta a
tc g g a tc g ttg c c g c a c tg a a c a g c g c c c c g ttc c a g g tc g c c tc c a ttt
a tg c g g c tg a a c cg a g g g a g a g c a g c ttta c g c c g tc tg g cc g c a g ttc g
c c c ttg g g cg a c
267
Appendix C
Table C .l List o f E. coli isolates collected from Tripoli and Benghazi

E. coli strain Site o f collection Place of collection
AES 11 Urine Al-Jamhoryia hospital
AES35 Floor o f toilet Al-Jamhoryia hospital
(ICU)
AES 5 8 Blood Al-Jamhoryia hospital
AES 120 Wall o f ICU Al-Jamhoryia hospital
AES202 Urine Al-Jamhoryia hospital
AES212 Swab from Benghazi Paediatric hospital
incubator
AES224 Floor o f ICU Benghazi Paediatric hospital
AES226 Urine Benghazi Paediatric hospital
AES227 Wall o f ICU Benghazi Paediatric hospital
AES230 Bed side in ICU Benghazi Paediatric hospital
AES231 Comer in ICU Benghazi Paediatric hospital
AES239 Wall o f ICU Benghazi Paediatric hospital
AES240 Corridor o f ICU Benghazi Paediatric hospital
AES248 Blood 7th o f October hosital
AES262 Pus 7th of October hospital
AES101 Floor o f ICU Al-Jamhoryia hospital
AES922 Urine Al-Jalla hospital Tripoli
AES932 Urine Maternity hospital Tripoli
AES937 Blood Bum and plastic surgery Tripoli
AES938 Floor o f ICU Al-Jalla hospital Tripoli
AES941 Wall o f ICU Al-Jalla hospital Tripoli
AES944 Floor o f toilet Bum and plastic surgery Tripoli
AES962 Urine Bum and plastic surgery Tripoli
AES964 Urine Bum and plastic surgery Tripoli
AES966 Floor o f ICU Maternity hospital Tripoli
AES971 Bedside Maternity hospital Tripoli
AES979 Urine Maternity hospital Tripoli
AES 1006 Blood Bum and plastic surgery Tripoli
AES 1037 Urine Bum and plastic surgery Tripoli
268
Figure C .l DNA sequence of A / a c t x - m - i s amplified from E. coli isolate
AES226
NNNNNNNNNNNNNNN ANNNNN GAN CN G A AT CN GCGGCGC ACGAT

CTTTT GGCCN GAT C AC
CGCG AT AT CGTT GGT GGT GCC AT AGCC ACCGCT GCCGGTTTT AT CC
CCCACAACCCAGGA
AGC AGGC AGTCC AGCCT G AAT GCTCGCT GC ACCGGT GGT ATT GCCT
TTCATCCATGTCAC
C AGCTGCGCCCGTT GGCT GT CGCCC AAT GCTTT ACCC AGCGT CAG A
TTCCGCANAGTTTG
CGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGAAT
GGCGGTGTTTAACGT
CGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGTCGG
GCG AACGCGGT GAC
GCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACG
TTATCGCTGTACTG
TAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACG
T GCTTTT CCGC AAT
CGG ATT AT AGTT AAC AAGGT C AG ATTTTTT GATCTC AACTCGCTGAT
TTAACAGATTCGG
TTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTAC
T GGT GCT GC AC AT
CGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCT
GT GTT AAT C AAT GC
CACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTT
GCTGTACGTCCGC
CGTTT GCGC AT AC AGCGGC AC ACTTCCT AAC AAC AGCGT GACGGTT
GCCGTCGCCATCNG
CGTGAACTGGCGCAGTGATTTTTTTAACCATGGGATTCCTTATTCTG
GA AG AT ACN A AAT
AACNACANNATGAATANNCCCCNANNNNCNCNNGNGNTTTTTNAT
TNNNNTT C ANNNN CN
NNNNNNNNC A A AGN ANNTNNNNNN GNNN CN
269
Figure C.2 Alignment of DNA sequence from figure C .l with DNA
> gb |H Q 2140 4 5 . 1 1 E n t e r o b a c t e r a e r o g e n e s s t r a i n K -307 p la sm id

in s e r t io n seq u en ce
IS E c p l TnpA IS E c p l (tnpA ) g e n e , c o m p le te c d s ; and b e t a - la c t a m a s e
CTX-M-15 (blaCTX-M -15) and h y p o t h e t i c a l p r o t e i n g e n e s ,
c o m p le te c d s
L en g th = 2 9 4 3
S c o r e = 1496 b i t s ( 8 1 0 ) , E x p e c t = 0 .0
I d e n t i t i e s = 8 2 1 /8 3 0 (99% ), Gaps = 1 /8 3 0 (0%)
Q uery 27
GAATCNGCGGCGCACGATCTTTTGGCCNGATCACCGCGATATCGTTGGTGGTGCCATAGC 86
S b jc t 2468
GAATCAGCGGCGCACGATCTTTTGGCCAGATCACCGCGATATCGTTGGTGGTGCCATAGC 24 09
Q uery 87
CACCGCTGCCGGTTTTATCCCCCACAACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCG 14 6
S b jc t 2408
CACCGCTGCCGGTTTTATCCCCCACAACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCG 2349
Q uery 147
CTGCACCGGTGGTATTGCCTTTCATCCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCA 206
S b jct 2348
CTGCACCGGTGGTATTGCCTTTCATCCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCA 228 9
Q uery 207
ATGCTTTACCCAGCGTCAGATTCCGCANAGTTTGCGCCATTGCCCGAGGTGAAGTGGTAT 266
S b jc t 2288
ATGCTTTACCCAGCGTCAGATTCCGCAGAGTTTGCGCCATTGCCCGAGGTGAAGTGGTAT 222 9
Q uery 26 7
CACGCGGATCGCCCGGAATGGCGGTGTTTAACGTCGGCTCGGTACGGTCGAGACGGAACG 3 26
S b jc t 2228
CACGCGGATCGCCCGGAATGGCGGTGTTTAACGTCGGCTCGGTACGGTCGAGACGGAACG 216 9
Q uery 327
TTTCGTCTCCCAGCTGTCGGGCGAACGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAA 3 86
S b jc t 2168
TTTCGTCTCCCAGCTGTCGGGCGAACGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAA 2109
Q uery 387
TCAGCTTATTCATCGCCACGTTATCGCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCA 44 6
S b jc t 2108
TCAGCTTATTCATCGCCACGTTATCGCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCA 204 9
270
Q uery 44 7
GTGACATCGTCCCATTGACGTGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATT 506
S b jc t 2048
Q uery 507
TTTTGATCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCG 566
S b jc t 1988
TTTTGATCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCG 192 9
Q uery 567
CGGCCGCGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAA 626
S b jc t 1928
CGGCCGCGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAA 186 9
Q uery 62 7
GTATTTGCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCC 686
S b jc t 1868
GTATTTGCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCC 180 9
Q uery 6 87
GCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTC 74 6
S b jc t 1808
GCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTC 174 9
Q uery 74 7
CTAACAACAGCGTGACGGTTGCCGTCGCCATCNGCGTGAACTGGCGCAGTGAt. 111111A 806
S b jc t 1748
CTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGTGATTTTTT-A 1690
Q uery 807 ACCATGGGATTCCTTATTCTGGAAGATa c n a a a t a s c n a ca n n a tg a a t a 856

I I II I I I I II II I I II I I II I II I I I I I I I II I I II III I II I I I I
S b jc t 168 9 ACCATGGGATTCCTTATTCTGGAAGATACGAAATAACAACAACATGAATA 164 0
> g b |H Q 2 1 4 0 4 4 . 1 | E n t e r o b a c t e r c lo a c a e s t r a i n K-221 p la sm id
I n c F : : FIB i n s e r t i o n
s e q u e n c e IS E c p l TnpA IS E c p l (tnpA) g e n e , c o m p le te c d s ; and
b e t a - l a c t a m a s e CTX-M-15 (blaCTX-M -15) and h y p o t h e t i c a l p r o t e in
g e n e s , c o m p le te c d s
L en gth = 2943
S c o r e = 1496 b i t s ( 8 1 0 ) , E x p e c t = 0 .0
I d e n t i t i e s = 8 2 1 /8 3 0 (99% ), Gaps = 1 /8 3 0 (0%)
Q uery 2 7
GAATCNGCGGCGCACGATCTTTTGGCCNGATCACCGCGATATCGTTGGTGGTGCCATAGC 86
S b jc t 2468
GAATCAGCGGCGCACGATCTTTTGGCCAGATCACCGCGATATCGTTGGTGGTGCCATAGC 24 09
271
Q uery 87
CACCGCTGCCGGTTTTATCCCCCACAACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCG 1 46
S b jc t 2408
CACCGCTGCCGGTTTTATCCCCCACAACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCG 2349
Q uery 14 7
S b jc t 2348
Q uery 2 07
ATGCTTTACCCAGCGTCAGATTCCGCANAGTTTGCGCCATTGCCCGAGGTGAAGTGGTAT 266
S b jc t 2288
ATGCTTTACCCAGCGTCAGATTCCGCAGAGTTTGCGCCATTGCCCGAGGTGAAGTGGTAT 2229
Q uery 267
CACGCGGATCGCCCGGAATGGCGGTGTTTAACGTCGGCTCGGTACGGTCGAGACGGAACG 326
S b jc t 2228
CACGCGGATCGCCCGGAATGGCGGTGTTTAACGTCGGCTCGGTACGGTCGAGACGGAACG 2169
Q uery 327
S b jc t 2168
Q uery 387
TCAGCTTATTCATCGCCACGTTATCGCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCA 446
S b jc t 2108
TCAGCTTATTCATCGCCACGTTATCGCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCA 2049
Q uery 44 7
S b jc t 2048
Q uery 507
S b jc t 1988
Q uery 567
S b jc t 1928
Q uery 62 7
272
S b jc t 1868
Q uery 6 87
GCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTC 746
S b jc t 1808
GCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTC 1749
Q uery 74 7
CTAACAACAGCGTGACGGTTGCCGTCGCCATCNGCGTGAACTGGCGCAGTGAt 11111: t A 806
S b jc t 1748
CTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGTGATTTTTT - A 1690
Q uery 807 ACCATGGGATTCCTTATTCTGGAAGATc tataacnac ta 856
273
Figure C.3 DNA sequence o f b la c tx-m-is amplified from E. coli isolate
AES228
GCGGCGCACGATCTTTTGGCCNGATCAC
CGCGATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTA
TCCCCCACAACCCAGGA
AGCAGGCAGTCCAGCCTGAATGCTCGCTGCACCGGTGGTATTG
CCTTTCATCCATGTCAC
CAGCTGCGCCCGTTGGCTGTCGCCCAATGCTTTACCCAGCGTC
AGATTCCGCANAGTTTG
CGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGA
ATGGCGGTGTTTAACGT
CGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGT
CGGGCGAACGCGGTGAC
GCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCC
ACGTTATCGCTGTACTG
TAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTG
ACGT GCTTTT CCGC AAT
CGG ATT AT AGTT AAC AAGGT C AG ATTTTTT GAT CTC AACTCGCT
GATTTAACAGATTCGG
TTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTT
T ACT GGT GCT GCACAT
CGC AAAGCGCTC AT CAGC ACG AT AAAGT ATTTGCG A ATT ATCT
GCTGTGTTAATCAATGC
CACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGT
TTTTGCTGTACGTCCGC
CGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACG
GTTGCCGTCGCCATCNG
CGTGAACTGGCGCAGTGATTTTTTTAACCATGGGATTCCTTATT
CTGGAAGATACNAAAT
AACN AC ANN AT G AAT ANNCCCCN ANNNN CN CNN GN GNTTTTT
Figure C.4 Alignment o f DNA sequence from figure C.3 with DNA
g b |J N 7 8 8 2 6 7 . 1 [ A c i n e t o b a c t e r b a u m a n n ii s t r a i n H I
h y d r o x y is o u r a te h y d r o la s e g e n e ,
c o m p le te c d s ; d is r u p t e d p y r im id in e u t i l i z a t i o n tr a n s p o r t e r
g e n e , p a r t ia l se q u e n c e ; in s e r t io n seq u en ce IS E cp l tr a n s p o sa se
( t n p A ) g e n e , c o m p l e t e c d s ; C T X -M 1 5 ( b la C T X - M 1 5 ) g e n e ,
c o m p le te c d s ; d is r u p t e d o r f4 7 7 g e n e , p a r t i a l s e q u e n c e ;
tr a n sp o so n
Tn3 tn p A g e n e , p a r t i a l s e q u e n c e ; a n d h y p o t h e t i c a l p r o t e i n
g e n e , c o m p le te c d s
L e n g th = 5 2 2 4
S core = 1489 b it s (8 0 6 ), E xpect = 0 .0

I d e n t i t i e s = 8 1 6 /8 2 4 (9 9 % ), G a p s = 1 /8 2 4 (0%)
274
Q uery 1
GCGGCGCACGATCTTTTGGCCNGATCACCGCGATATCGTTGGTGGTGCCATAGCCACCGC
60
S b jc t 3516
GCGGCGCACGATCTTTTGGCCAGATCACCGCGATATCGTTGGTGGTGCCATAGCCACCGC
3457
Q uery 61
TGCCGGTTTTATCCCCCACAACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCGCTGCAC
120
S b jc t 3456
TGCCGGTTTTATCCCCCACAACCCAGGAAGCAGGCAGTCCAGCCTGAATGCTCGCTGCAC
3397
Q uery 121
CGGTGGTATTGCCTTTCATCCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCAATGCTT
180
S b jc t 3396
CGGTGGTATTGCCTTTCATCCATGTCACCAGCTGCGCCCGTTGGCTGTCGCCCAATGCTT
3337
Q uery 181
TACCCAGCGTCAGATTCCGCANAGTTTGCGCCATTGCCCGAGGTGAAGTGGTATCACGCG
240
S b jc t 3336
TACCCAGCGTCAGATTCCGCAGAGTTTGCGCCATTGCCCGAGGTGAAGTGGTATCACGCG
3277
Q uery 241
GATCGCCCGGAATGGCGGTGTTTAACGTCGGCTCGGTACGGTCGAGACGGAACGTTTCGT
300
S b jc t 3276
GATCGCCCGGAATGGCGGTGTTTAACGTCGGCTCGGTACGGTCGAGACGGAACGTTTCGT
3217
Q uery 3 01
CTCCCAGCTGTCGGGCGAACGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAATCAGCT
360
S b jc t 3216
CTCCCAGCTGTCGGGCGAACGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAATCAGCT
3157
Q uery 361
TATTCATCGCCACGTTATCGCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACA
420
275
S b jc t 3156
TATTCATCGCCACGTTATCGCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACA
3097
Q uery 421
TCGTCCCATTGACGTGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGA
480
S b jc t 3096
TCGTCCCATTGACGTGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGA
3037
Q uery 4 81
TCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCG
540
S b jc t 3036
TCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCG
2977
Q uery 541
CGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTT
600
S b jc t 2976
CGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTT
2917
Q uery 601
GCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTA
660
S b jc t 2916
GCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTA
2857
Q uery 661
ATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACA
720
S b jc t 2856
ATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACA
2797
Q uery 721
ACAGCGTGACGGTTGCCGTCGCCATCNGCGTGAACTGGCGCAGTGAt 1 1 1 1 ; 11AACCATG
780
S b jc t 2796
ACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGTGATTTTTT-AACCATG
2738
Q uery 781 GGATTCCTTATTCTGGAAGATACNAAATAACNACANNATGAATA 824

I I I I I I I I I I I I I II I I I I I I I I M M : I 1 III l l l l l l l
S b jc t 2 73 7 GGATTCCTTATTCTGGAAGATACGAAATAACAACAACATGAATA 2 694
276
Figure C.5 D NA sequence o f ^/actx-m-15 amplified from E. coli isolate
AES232
CGCG AT AT CGTT GGT GGT GCC AT AGCC ACCGCT GCCGGTTTT AT CC

CCCACAACCCAGGA
AGC AGGC AGTCC AGCCT GA AT GCTCGCT GC ACCGGT GGT ATT GCCT
TTCATCCATGTCAC
C AGCT GCGCCCGTT GGCTGT CGCCC AAT GCTTT ACCC AGCGT CAGA
TTCCGCANAGTTTG
CGCC ATT GCCCG AGGT GA AGT GGT AT C ACGCGG ATCGCCCGG A AT
GGCGGTGTTTAACGT
CGGCTCGGT ACGGTCG AG ACGG AACGTTT CGTCTCCC AGCTGTCGG
GCGAACGCGGTGAC
GCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACG
TTATCGCTGTACTG
TAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACG
TGCTTTTCCGCAAT
C GG ATT AT AGTT AAC AAGGT C AG ATTTTTT G ATCT CA ACTCGCT GAT
TTAACAGATTCGG
T GGT GCT GC AC AT
CGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCT
GT GTT AAT C A AT GC
GCTGTACGTCCGC
CGTTT GCGC AT AC AGCGGC AC ACTTCCTAAC AAC AGCGT GACGGTT
GCCGTCGCCATCNG
CGT GAACT GGCGC AGT G ATTTTTTT AACC AT GGGATTCCTT ATT CTG
GAAG AT ACN AAAT
AACNACANNATGAATANNCCCCNANNNNCNCNNGNGNTTTTTNNN
NNN
emblFR828676.1| E s c h e r i c h i a c o l i p la s m id pC T X 913 tn p A gene,

b la C T X -M -1 5 g e n e
a n d d e l t a tn p A g e n e ( p a r t i a l ) , i s o l a t e 9 1 3
L e n g th = 2 6 5 6
S core = 1441 b it s (7 8 0 ), E xpect = 0 .0

I d e n t i t i e s = 7 8 9 /7 9 6 (9 9 % ), G a p s = 1 /7 9 6 (0% )
Q uery 1
CGCGATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTATCCCCCACAACCCAGGA
60
277
S b jc t 1674
CGCGATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTATCCCCCACAACCCAGGA
1615
Q uery 61
AGCAGGCAGTCCAGCCTGAATGCTCGCTGCACCGGTGGTATTGCCTTTCATCCATGTCAC
120
S b jc t 1614
AGCAGGCAGTCCAGCCTGAATGCTCGCTGCACCGGTGGTATTGCCTTTCATCCATGTCAC
1555
Q uery 121
CAGCTGCGCCCGTTGGCTGTCGCCCAATGCTTTACCCAGCGTCAGATTCCGCANAGTTTG
180
S b jc t 1554
CAGCTGCGCCCGTTGGCTGTCGCCCAATGCTTTACCCAGCGTCAGATTCCGCAGAGTTTG
1495
Q uery 181
CGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGAATGGCGGTGTTTAACGT
240
S b jc t 1494
CGCCATTGCCCGAGGTGAAGTGGTATCACGCGGATCGCCCGGAATGGCGGTGTTTAACGT
1435
Q uery 241
CGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGTCGGGCGAACGCGGTGAC
300
S b jc t 1434
CGGCTCGGTACGGTCGAGACGGAACGTTTCGTCTCCCAGCTGTCGGGCGAACGCGGTGAC
1375
Q uery 3 01
GCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACGTTATCGCTGTACTG
360
S b jc t 1374
GCTAGCCGGGCCGCCAACGTGAGCAATCAGCTTATTCATCGCCACGTTATCGCTGTACTG
1315
Q uery 3 61
TAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTTTTCCGCAAT
420
S b jc t 1314
TAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCCATTGACGTGCTTTTCCGCAAT
1255
Q uery 421
CGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACAGATTCGG
480
278
S b jc t 1254
CGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACAGATTCGG
1195
Q uery 4 81
TTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGCTGCACAT
540
S b jc t 1194
TTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGCTGCACAT
1135
Q uery 541
CGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAATCAATGC
600
S b jc t 1134
CGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAATCAATGC
1075
Q uery 601
CACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGC
660
S b jc t 1074
CACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGC
1015
Q uery 661
CGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCGCCATCNG
720
S b jc t 1014
CGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCGCCATCAG
955
Q uery 721
CGTGAACTGGCGC A G T G A 1 1 1 1 1 1 1 A A C CATGGGATTCCTTATTCTGGAAGATa c n a a a t
780
S b jc t 954 CGTGAACTGGCGCAGTGATTTTTT-
AAC CATGGGATT C CTTATTC TGGAAGATACGAAAT 8 96
Q uery 781 aacnacannatgaata. 7 96

III III l l l l l l l
S b jc t 8 95 AACAACAACATGAATA 880
279
Figure C.7 DNA sequence o f blacix-u -3 amplified from E. coli isolate
AES228 amplified by CTX-M-F
NNNNNNNNN CNNNN GCN GTT GTT AGG AGT GT GCCGCT GT AT GCGC

AAACGGCGGACGTAC
AGC AA A A ACTT GCCG AATT AG AGCGGC AGTCGGGAGGC AG ACT GG
GT GT GGC ATT GATT A
AC AC AGC AG AT AATT CGC AAAT ACTTT AT CGT GCT GAT G AGCGCTT
TGCGATGTGCAGCA
CC AGT AAAGT GAT GGCCGCGGCCGCGGT GCT GAAG AA AAGT G AAA
GCGAACCGAATCTGT
T AAAT C AGCG AGTT GAG AT CAAA AAAT CT GACCTT GTT AACT AT AA
TCCGATTGCGGAAA
AGC ACGT C AAT GGG ACG AT GT C ACT GGCT GAGCTT AGCGCGGCCG
CGCTACAGTACAGCG
AT AACGT GGCG AT G AAT AAGCT GATT GCT C ACGTT GGCGGCCCGGC
TAGCGTCACCGCGT
TCGCCCGACAGCTGGGAGACGAAACGTTCCGTCTCGACCGTACCGA
GCCGACGTTAAACA
CCGCCATTCCGGGCGATCCGCGTGATACCNCTTCACCTCNGGCAAT
GGCGCANANTCTGC
GGAATCTGACGCTGGGNAANGNNTNGGGCGACNNCNNACNGGCGC
NNCTGGTGANN
280
Figure C.8 Alignment o f DNA sequence from figure C.7 with DNA sequence
from gene bank
g b ] H Q 2 1 4 0 5 2 .1 1 E n t e r o b a c t e r c lo a c a e s t r a i n S -4 4 0 p la s m id
I n c F : : F IB i n s e r t i o n
s e q u e n c e I S E c p l , p a r t i a l s e q u e n c e ; a n d b e t a - l a c t a m a s e CTX-M-3
(b la C T X -M -3 ) a n d h y p o t h e t i c a l p r o t e i n g e n e s , c o m p l e t e c d s
L e n g th = 1 4 9 8
S c o r e = 1088 b i t s (5 8 9 ), E x p ect = 0 .0
I d e n t i t i e s = 6 0 0 / 6 1 1 (9 8 % ), G a p s = 0 / 6 1 1 (0%)
Q uery 17
AGCTCNGCCNGTGACNTCGTCCCNTTGACGTGCTTTTCCGCAATCGGATTATAGTTAACA 76
S b jc t 613
AGCTCAGCCAGTGACATCGTCCCATTGACGTGCTTTTCCGCAATCGGATTATAGTTAACA 554
Q uery 77
AGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTC 13 6
S b jc t 553
AGGTCAGATTTTTTGATCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTC 4 94
Q uery 13 7
TTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCA 196
S b jc t 493
TTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCA 434
Q uery 197
GCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCT 256
S b jc t 433
GCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCT 3 74
Q uery 257
CCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGC 316
S b jc t 373
CCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGC 314
Q uery 317
GGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGT 3 76
S b jc t 313
GGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGT 254
Q uery 3 77
GATTTTTTAACCATGGGATTCCTTATTCTGGAAGAGACGAAATAACAACAACATGAATAG 43 6
S b jc t 253
GATTTTTTAACCATGGGATTCCTTATTCTGGAAGAGACGAAATAACAACAACATGAATAG 194
Q uery 43 7
TCAATATTTTACCTGAAGCGAGCCACAACGCGTCCGATTTTATGCTTCCGAAAGGCAAAT 4 96
281
S b jc t 193
TCAATATTTTACCTGAAGCGAGCCACAACGCGTCCGATTTTATGCTTCCGAAAGGCAAAT 134
Q uery 4 97
ACNGACGTCGCCAGAATGAAACCTAAATTCCACGTGTGTTTTTTATTANCTnnnanaaTC 556
S b jc t 133
ACGGACGTCGCCAGAATGAAACCTAAATTCCACGTGTGTTTTTTATTAGCTTCAAAAATC 74
Q uery 55 7
ACTATTTCACGAAGAATTTAGACTGCTTCTCACACATTGTAACATTATTTACAACCNCCT 616
S b jc t 73
ACTATTTCACGAAGAATTTAGACTGCTTCTCACACATTGTAACATTATTTACAACCACCT 14
Q uery 617 TTCAATCATTT 62 7

I! I I I I II 11 I
S b jc t 13 TTCAATCATTT 3
Figure C.9 DNA sequence of blacix-u-3 amplified from E. coli isolate

CN GT G AC AT CGT CCCNTT G ACGT GCTTTTCCGC AAT

CGG ATT AT AGTT AAC AAGGT CAG ATTTTTT GAT CTC AACTCGCT GAT
TTAACAGATTCGG
T GGT GCT GC AC AT
CGC AAAGCGCTC AT C AGC ACGAT AAAGT ATTT GCGAATTATCT GCT
GTGTTAATCAATGC
GCTGTACGTCCGC
CGTTT GCGC AT AC AGCGGC AC ACTTCCTAAC AAC AGCGT GACGGTT
GCCGTCGCCATCAG
CGT G AACT GGCGC AGT GATTTTTT AACC AT GGG ATTCCTT ATTCTGG
AAGANACGAAATA
ACAACAACATGAATAGTCNATATTTTACCTGAGGGGAGNCAANNN
NCNTCNNANTTNANG
CTTCCGAAAGGAAAATACAGAGNTCNNCANAANGAAANNNAATAT
NNACNNGTGNNTTTN
ANNNNNNTTNT A A A AT C ACT ATTT CACGA AGAATTTAG ACT GCTT C
T C AC AC ATT GT AAC
ATTATTTACAACCACCTTTCNNNNNNNNNNNNNNNNNNNGNNNNG
NNNNNCNNANNNNNN
CNNNNNNNN CNN C CNTT GTNNT CNTTTN C GC ANNN GNNTG ACN CA
CTCNCNNANTANNNNANNNNNNNCNTTTCCTTTTNTNNNNNNCGN
GNNCGNNN
282
NNNNCN
sequence from gene bank
E s c h e r i c h i a c o l i s t r a i n S - 7 4 1 p l a s m i d I n c L /M i n s e r t i o n s e q u e n c e
I S E c p l , p a r t i a l s e q u e n c e ; b e t a - l a c t a m a s e C T X -M -3 ( b l a C T X - M - 3 )
a n d h y p o t h e t i c a l p r o t e i n g e n e s , c o m p l e t e c d s ; a n d M ucA
(m u c A ) g e n e , p a r t i a l c d s
L e n g th = 2 02 8
Score = 907 b i t s (4 9 1 ), E xpect = 0 .0

I d e n t i t i e s = 5 5 2 /5 9 7 (9 2 % ), G a p s = 7 /5 9 7 (1%)
Q uery 3
GTGACATCGTCCCNTTGACGTGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATT
62
S b jc t 603
GTGACATCGTCCCATTGACGTGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATT
544
Q uery 63
TTTTGATCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCG
122
S b jc t 543
TTTTGATCTCAACTCGCTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCG
484
Q uery 12 3
CGGCCGCGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAA
182
S b jc t 483
CGGCCGCGGCCATCACTTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAA
424
Q uery 183
GTATTTGCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCC
242
S b jc t 423
GTATTTGCGAATTATCTGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCC
364
Q uery 24 3
GCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTC
302
283
S b jc t 363
GCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTC
304
Q uery 3 03
CTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGTGATTTTTTAA
362
S b jc t 303
CTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGTGATTTTTTAA
244
Q uery 3 63
CCATGGGATTCCTTATTCTGGAAGANACGAAATAACAACAACATGAATAGTCNATATTTT
422
S b jc t 243
CCATGGGATTCCTTATTCTGGAAGAGACGAAATAACAACAACATGAATAGTCAATATTTT
184
Q ue r y 423 ACCTGAGGGGAGnc a -
a n n n n c n tc n n a n ttn a n g c ttc c g a a a g c r a a a a ta c a g a -c m t 4 80
m i n i i i i i ii r i ii i ii i iiii i i i i i i i i
i i i i i i ii i i
S b jc t 183 ACCTGAAGCGAGCCACAACG-
CGTCCGATTTTATGCTTCCGAAAGGCAAATACGGACG- T 12 6
Q uery 4 81 c n n c a n a a n g a a a n n n a a ta tn n -
a c r m g tg r m tttn a n n n n n n ttn ta a a a T C A C T A T T 53 9
S b jc t 125 CGCCAGAATGAAACCTAA-ATTCCACGTGTGTTTTTTATTAGC-
TTCAAAAATCACTATT 68
Q uery 54 0
TCACGAAGAATTTAGACTGCTTCTCACACATTGTAACATTATTTACAACCACCTTTC 596
S b jc t 67
TCACGAAGAATTTAGACTGCTTCTCACACATTGTAACATTATTTACAACCACCTTTC 11
284
Figure C .ll DNA sequence o f blacxx-u-3 amplified from E. coli isolate
NNNNNNNNNNNGCGCNANCTNNGCCNGTGACNTCGTCCCNTTGAC
GTGCTTTTCCGCAAT
CGG ATT AT AGTT AAC AAGGT CAG ATTTTTT GAT CT C AACTCGCT GAT
TTAACAGATTCGG
T GGT GCT GC AC AT
CGC A A AGCGCTC AT C AGC ACGAT AAAGT ATTT GCG AATT ATCT GCT
GT GTT AAT C A AT GC
GCTGTACGTCCGC
CGTTT GCGC AT AC AGCGGC AC ACTTCCT AAC AAC AGCGT GACGGTT
GCCGTCGCCATCAG
CGT GAACT GGCGC AGT GATTTTTT AACC AT GGG ATTCCTT ATT CT GG
AAGANACGAAATA
AC AAC A AC AT GAAT AGT CN AT ATTTT ACNT GANN GNN GN CNNNNN
NCNNCNNANTTNATG
CTTCCNAAAGGAAAATANANNNNTNNNCNGAANGNNNNNNANNN
NNNACNNGNGNNNTTA
NNNNNNNTTTN AA A ATC ACT ATTT C ACG A AG AATTT AG ACT GCTTC
TCACACATTGNAAC
NNNNNTTNNNAACCNCCTTTNNNNNNNNTTNNNNNNANNNNGGGA
N CNN GNN CNNN A ANN
NNNNN CNNN GNNNT CNTN C CNNTNNNNT GCTTTT CN GC A AT CN GA
TT AT ANTTT ANNN GG
N CNN ANTTNTT GAN CN CNNT CN CNNNNNN ANNN AATTNN GNNT CN
NTTNCTTTTNNNTCN
NNNCNNNNNCCNNNNNCNTNCTTTNNNGNNNCTNNNCATNCAANN
NNCTCNT CNNNN CAT
NANNNNNNNNNANTNTCNGCNGNNNTTAANNANGNNNNNNNNNG
NNNCCNANGNNNNNNA
ANTNNN GC ANNNTTTTTNNNNNNNNNNN GNNNNNN
Figure C.12 Alignment o f DNA sequence from figure C.l 1 with DNA
sequence from gene bank
gb|GQ292713.11 K le b s ie lla p n e u m o n ia e s tr a in S -3 3 4 p la s m id I n c L /M
in s e r tio n seq u en ce
I S 2 6 t r a n s p o s a s e tn p A I S 2 6 (tn p A ) g e n e , c o m p le t e c d s ;
i n s e r t i o n s e q u e n c e I S E c p l, c o m p le te s e q u e n c e ; b e t a - la c t a m a s e
C T X -M -3 ( b l a C T X - M - 3 ) g e n e , c o m p l e t e c d s ; M ucA (m u cA ) g e n e ,
p a r t i a l c d s ; and unknow n g e n e
L e n g th = 3 2 6 0
Score = 747 b i t s (404), E x p e c t = 0 .0

I d e n titie s = 407/410 (99%), G aps = 0 / 4 1 0 (0%)
285
Strand=Plus/Minus
Q uery 1
GACGTGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCG
60
Sbjct 1803
GACGTGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCG
1744
Q uery 61
CTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCAC
120
Sbjct 1743
CTGATTTAACAGATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCAC
1684
Q uery 121
TTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATC
180
Sbjct 1683
TTTACTGGTGCTGCACATCGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATC
1624
Q uery 181
TGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAG
240
Sbjct 1623
TGCTGTGTTAATCAATGCCACACCCAGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAG
1564
Q uery 241
TTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGAC
300
Sbjct 1563
TTTTTGCTGTACGTCCGCCGTTTGCGCATACAGCGGCACACTTCCTAACAACAGCGTGAC
1504
Q uery 301
GGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGTGATTTTTTAACCATGGGATTCCTTAT
360
Sbjct 1503
GGTTGCCGTCGCCATCAGCGTGAACTGGCGCAGTGATTTTTTAACCATGGGATTCCTTAT
1444
Q uery 3 61 TCTGGAAGANACGAAATAACAACAACATGAATAGTCNATATTTTACNTGa
410
M I N I M I 1 1 1 1 1 1 1 I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I l l l l l l l l l III
Sbjct 1443 TCTGGAAGAGACGAAATAACAACAACATGAATAGTCAATATTTTACCTGA
1394
286
Appendix D
TABLE D .l Ceftazidime resistant Gram-negative bacteria isolated from

Hospital environmental swabs.
Swab Bacterial isolate Location

301 Achrom obacter sp Tripoli central hospital
302 Pseudomonas putida Gergarish
303 Aerom onas caviae 1st of September
304 Achrom obacter sp. Andalus
305 Acinetobacter baumannii Gergarish
306 Stenotrophomonas maltophilia Omar Mokhtar
307 Pseudomonas pseudoalcaligenes Omar Mokhtar
308 Stenotrophomonas maltophilia Seraj
309 Achrom obacter sp. Seraj area
310 Achrom obacter sp Siahia
311 Pantoe a agglomerans Seraj
312 Pseudomonas aeruginosa Siahia
313 Achrom obacter sp Omar Mokhtar
314 Tatumella ptyseos Siahia
315 Pseudom onas putida Seraj
316 Pseudom onas putida Omar Mokhtar
317 Achrom obacter sp Omar Mokhtar
318 Burkholderia cepacia/RaXstonm pickettii Seraj

319 Achrom obacter sp Seraj
320 Pseudom onas putida Gergarish
321 Pseudom onas putida Seraj
322 Stenotrophomonas maltophilia Seraj
323 Tatumella ptyseos Gergarish

325 Enterobacter cloacae Omar Mokhtar
326 Citrobacter freundii Seraj

327 Tatumella ptyseos Seraj
287
328 Pantoea agglomerans Jamahiyria
329 Achrom obacter sp Jamahiyria
330 Achrom obacter sp Jamahiyria
331 Ochrobactrum anthropi Jamahiyria
332 P. aeruginosa Serah
335 Acinetobacter baumannii Gergarish
336 Leclercia adecarboxyalata Seraj
337 Stenotrophomonas maltophilia Akhadra
338 Enterobacter cloacae Alkhadra
288
Figure D .l Hydrolysis of antibiotic meropenem by TMB-1
Vo #1 Vo Vo # 3 [E]: 100nM
VMAX 6.102 26.42
KM 355.2 75.11
Meropenem
30n
■ Vo #1
• Vo #3 [E]: 100nM
o
>
0 100 200 300 400 500 600 700 800 9001000

[S] (J.M
289
Figure D.2 Hydrolysis of antibiotic Ertapenem by TMB-1
VMAX 15.47
KM 31.06
Ertapenem
17.5-1
Vo #1 [E]: 100nM
15.0-
12.5-
o 10.0-
>
7.5-
5.0
2.5
o.o-
0 100 200 300 400 500 600 700 800 9001000
[S] pM
290
Figure D.3 Hydrolysis of antibiotic ceftazidime by TMB-1
Vo #1 [E]: 1 Vo #3 [E]: 100nM Vo #3 [E]: 1 (iM

VMAX 1.927 2.632 2.577
KM 18.89 187.6 91.81
Ceftazidime
2.5
■ Vo #1 [E : 1|iM
2.0 a Vo #3 [E : 100nM
• Vo #3 [E : 1|iM
1.5*
o
>
1 . 0-
0.5*
0.0
0 50 100 150 200 250 300
[S] jiM
291
Figure D.4 Hydrolysis of antibiotic ampicilin by TMB-1
Vo #1 [E]: 100nM Vo #2 [E]: 100nM

VMAX 4 .4 9 0 5.822
KM 11.82 27.37
Ampicillin
5.5-i
5.0- ■ Vo #1 [E]: 100nM
4.5- ▼ Vo #2 [E]: 100nM
4.0-
3.5-
o 3.0-
> 2.5-
2 .0-
1.5-
1. 0-
0.5-
0 . 0-
0 25 50 75 100 125 150
[S] pM
292
Figure D.5 Hydrolysis of antibiotic imipenem by TMB-1
Vo #1 [E]: 10nM Vo #2 [E]: 10nM Vo #3 [E]:100nM

VMAX 21.07 11.60 35.30
KM 1909 614.0 200.8
Imipenem
35-1
■ Vo #1 [E]: 10nM
30-
A Vo #2 [E]: 10nM
25- • Vo #3 [E]:100nM
20-
15-
10-
0 250 500 750 1000 1250

[S] pM
293
Figure D.6 Hydrolysis of antibiotic cefoxitin by TMB-1
VMAX 4.037
KM 69.02
Cefoxitin
■ Vo #1 [E]: 100nM
o
>
100 200 300 400 500

[S] liM
294
Figure D.7 Hydrolysis of antibiotic cefuroxime by TMB-1
Vo #1 [E]: 1
VMAX 16.42
KM 8.560
Cefuroxime
20-1
Vo #1 [E]: VM
o
>
0 100 200 300 400 500 600
295
Figure D.8 Hydrolysis of antibiotic piperacillin by TMB-1
V o #1 [E]: 100nM
VMAX 6.831
KM 7 2 .1 3
Piperacillin
6.5-1
6 . 0- Vo #1 [E
5.5-
5.0-
4.5-
4.0-
O 3.5-
> 3.0-
2.5-
2 .0-
1.5-
1. 0 -
0.5-
0 .0- —r~ —
T—
100 200 300 400 500 600
[S] \M
296
Figure D.9 full class 1 integron (3kb) A/flTMB-b aac6II and blaoxx -4 from
Achromobacterxylosoxidans AES301
T AG AGG A AT AAT GG AAT GCG ACC ATTTTT ATTTTT AAT A ATTTTT AT

CAGTCATTTCGCTTTTGCCAACGAAGAAATACCCGGATTGGAAGTT
GAGG AA ATT GAC A ACGGCGTTTTTTT GC AC AAGT CAT AC AGCCGGG
T GG A AGGCTGGGGCCT GGT A AGTT CAA ACGG ACTT GTT GT CAT CAG
CGGCGG AA A AGC ATT C ATT ATT G AC ACT CC AT GGT C GG A AT C AG AT
AC AG AAAAGCTT GT AGATT GG AT ACGAT CAAAAAAGT AT GAGCT G
GCGGG AAGC ATTTCT AC AC ATT C AC ACG A AG AC A AG ACT GCCGGT
AT A AAAT GGCTAAACGGC AAAT CC ATT ACT AC AT AT GCCT CAGCGC
T G ACT AAT GA A ATT C TA A A A AG AG AGGGT AAGG AGC AGGC A AGG A
GCTC ATT CAAAGGT AAT G AATTTTCGCT GAT GGACGGTTTTCT AG A
AGTCTATT ATCCCGG AGGCGGCC AT ACT ATT GATAACTT AGT GGT A
T GG AT C C CT AGTT C A AAA AT ATT GT AT GGC GGCT GTTT CAT ACGT A
GCTT GGAATCC AGT GGGCT AGGTT AC ACT GGT GAAGCTAA AATT GA
TC AGT GGCC AC AATCCGCT AGAAAT AC AATTTCGAAGTATCCT GAA
GCT AAG ATT GT GGT GCCTGGT CAT GGAAA AATT GGCGATTT CG AGT
TGTTAAAACATACCAAGgTCcTTGCAGAAAaGGCCTCTAACAAGGCC
AATCACGGCGACCGCTGACGCGGCGCGTGTcgTTAGGCAGCACA
Gagcgaccatttcatgtccgcgagcaccccccccataactcttcgcctcatgaccgagcgcgacctgccgatg
ctccatgattggctcaaccggccgcacatcgttgagtggtggggtggtgacgaagagcgaccgactcttgatga
agtgctggaacactacctgcccagagcgatggcggaagagtccgtaacaccgtacatcgcaatgctgggcga
ggaaccgatcggctatgctcagtcgtacgtcgcgctcggaagcggtgatggctggtgggaagatgaaactgat
ccaggagtgcgaggaatagaccagtctctggctgacccgacacagttgaacaaaggcctaggaacaaggctt
gtccgcgctctcgttgaactactgttctcggaccccaccgtgacgaagattcagaccgacccgactccgaacaa
ccatcgagccatacgctgctatgagaaggcaggattcgtgcgggagaagatcatcaccacgcctgacgggcc
ggcggtttacatggttcaaacacgacaagccttcgagagaaagcgcggtgttgcctaacaactcattcaagccg
acgccgcttcgcggcgcggcttaattcaggtgttagccaagccgttaaaattaagccctttaccaaaccaataca
aaccaatacttgttatgaaaaacacaatacatatcaacttcgctatttttttaataattgcaaatattatctacagcagc
gccagtgcatcaacagatatctctactgttgcatctccattatttgaaggaactgaaggttgtt
TTTT ACTTT ACGAT GT AT CC AC A A ACGCT GAAATT GCTC A ATTC A AT

AAAGC AAAGT GT GC AACGC AAAtggcaccagattcaactttcaagatcgcattatcactT
AT GGC ATTT GAT GCGG AAAT AAT AG AT CAG AAAACc AT ATT C A A AT
GGGAtAAAACCCCCAAAGGAATGGAGATCTGGAACAGCAATCATA
CACC A AAG ACGT GG AT GC AATTTT CT GTT GTTT GGGTTTCGC A AG A
AAT AAC C CA A A A A ATT GG ATT AAAT A A A AT CRAG AATT AT CTC AA
AG ATTTT GATT AT GG A AAT CA AG ACTT CTCTGG AG At AA AG A A AG A
AAC A ACGG ATT A AC AG AAGC AT GGCTCG AAAGT AGCTT AAAA ATT
TCACC AG A AGA AC A AATT CAATTCCT GCGT AAA ATT ATT AAT C AC A
AT CTCCC AGTT A AAA ACT CAGCC AT AG AA AAC ACC AT AG AG A AC A
T GT AT CT AC AAGATCTGGAGAATAGT AC A AAACTGT AT GGGA A AA
CT GGT GC AGG ATTC AC AGC AAAT AG AACCTT AC AA AACGG AT GGT
TT G A AGGGTTT ATT AT A AGC A A AT C AGG AC AT AAAT AT GTTTTT GT
297
GTCCGC ACTT AC AGG AAACTT GGGGTCG A ATTT AAC AT CAAGC AT A
AA AGCC A AGAA AAAT GCGAT CACC ATT CT AAAC AC ACT AAATTT AT
A AAAAAT CT AAT GGC AAAAT CGCCC AACCCTT CAAT CAAGTCGGG
ACGGCCAAAAGCAAGCTTTTGGCTCCCCTCGCTGGCGCTCGGCGCC
CCTT ATTT C A A ACGTT AG AT GC ACT AAGC AC AT AATT GCT CAC AGC
CA A ACT AT C AGGTC AAGT CT GCTTTT ATT ATTTTT AAGCGT GC AT A A
T A AGCCCTAC AC A A ATT GGG AG AT AT ATC A
298
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Antibiotic Resistance Mechanisms in Gram-Negative Bacteria

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Antibiotic Resistance Mechanisms in Gram-Negative Bacteria

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CHARACTERISATION OF ANTIBIOTIC RESISTANCE

MECHANISMS IN GRAM-NEGATIVE BACTERIA FROM

Allaaeddin Ali El Salabi

A thesis submitted for the degree of Doctor of Philosophy

Cardiff University, School of Medicine, Department of

All rights reserved

INFORMATION TO ALL USERS

"i^m d ^ ^ / (candidate) Date

This thesis is the result of my own independent work/investigation, except

Signe d , ^ 7 .. (candidate) Date .. OT/j). /?:?. !!

I hereby give consent for my thesis, if accepted, to be available for

Signed (candidate) Date

As very little information is known of the antibiotic resistance in Gram-

negative bacteria in Libya in addition to the desperate need for insight

knowledge o f the antibiotic resistance in Libyan hospitals, this study was

undertaken to investigate the mechanism of antibiotic resistance in isolates

collected from clinical, non-clinical and environmental samples from Tripoli

bedsides, curtains, floors, toilets, workstations, mechanical ventilators,

clearly demonstrates the emergence of MDR Gram-negative bacteria in

clinical revealing the long standing infection control problem in these

hospitals. K. pneumoniae was found as the most frequently isolated strain

being disseminated in hospitals and outside hospitals followed by E. coli. K.

pneumoniae and E. coli were detected harbouring blactx-m group 1 in

association with ISEcpl the enhancer of the p-lactamase gene movement.

More importantly, ^/«ctx-m-is in association with ISEcpl were detected carried

on conjugative plasmids o f different sizes and able to move via Libyan K.

pneumoniae and E. coli to sensitive bacteria via conjugation. Some isolates of

K. pneumoniae were clonally related and were in some cases found in

types among K. pneumoniae were discovered in this study, one of which K.

Characterisation o f P. aeruginosa showed the emergence of clonally related

both strains. Interestingly, 6 /<zvim-2 was found chromosomally mediated

environmental bacteria A. xylosoxidans, it is moreover the first MBL

Presentations given from this study

1- Phenotypic and Genotypic Characterisation of Clinical and non-

2- Identification o f Tn402, Class 1 integrons and ISCR elements among

3- Novel subclass o f a Group B1 Metallo-p-lactamase, bla-\uB-u in

4- The tniC-like transposon Tn5090 is commonly found in Klebsiella

1- Salabi, A. E., M. A. Toleman, J. Weeks, T. Bruderer, R. Frei, and T. R.

2- Chouchani, C., R. Marrakchi, and A. El Salabi. 2011. Evolution of beta-

3- Chouchani, C., R. Marrakchi, L. Ferchichi, A. El Salabi, and T. R.

4- Chouchani, C., A. El Salabi, R. Marrakchi, L. Ferchichi, and T. R.

5- Allaaeddin El Salabi, Pardha Saradhi Borra, Mark A. Toleman, 0rjan

6- Allaaeddin El Salabi, Mark A. Toleman, Ahmed Matmati, Chedly

7- Allaaeddin El Salabi, Mark A. Toleman, Abdulazizi Zorgani and

8- Allaaeddin El Salabi, Mark A. Toleman, Asma Alramli and Timothy R.

To the staff at Reference Centre for Detection o f Antimicrobial Resistance, Department of

To Dr Amanda Tonks for her encouragement and support

To my friend Othman Boaisha for his kind encouragement and support

To staff o f Faculty o f Public Health, University o f Benghazi for their support

To Ranya, my wife for, her support, patience and endurance

the spirit o f my father, the

spirit o f my brother and

to the new Libya

Fig. 1.1 Chemical structure o f daptomycin.............................................. 6

Fig. 1 .2 Chemical structure o f oxazolidinone radezolid................... 6

Fig. 1.3 Chemical structure o f the fluoroquinolone delafloxacin 6

Fig. 1.4 Chemical structure o f the aminoglycoside ACHN-490 ....... 7

Fig. 1.5 Chemical structure o f the tetracycline om adacycline 7

Fig. 1.6 Chemical structure o f the Avibactam N X L -104....................... 7

Fig. 1.7 Cell wall envelopes o f Gram-positive and Gram-negative

Fig. 1.8 N-formimodoyl-thienamycin............................................... 15

Fig. 1.9 Inhibition o f protein synthesis by aminoglycosides 19

Fig. l.li Inhibition o f cell wall synthesis by p-lactam s.................... 20